Method for the treatment of neurological disorders by enhancing the activity of beta-glucocerebrosidase

ABSTRACT

Provided is a method of increasing the stability of wild-type β-glucocerebrosidase. Also provided are methods of treating and/or preventing an individual having a neurological disease in which increased expression or activity of β-glucocerebrosidase in the central nervous system would be beneficial. This method comprises administering an effective amount of a pharmacologic chaperone for β-glucocerebrosidase, with the proviso that the individual does not have a mutation in the gene encoding β-glucocerebrosidase. Further provided are β-glucocerebrosidase inhibitors which have been identified as specific pharmacologic chaperones and which have been shown to increase activity of β-glucocerebrosidase in vivo in the central nervous system.

This application claims priority from U.S. Provisional PatentApplication Ser. No. 60/815,952, filed on Jun. 23, 2006, the disclosureof which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to methods of increasing the activity of thelysosomal enzyme β-glucocerebrosidase for the treatment ofα-synucleinopathies such as Parkinson's disease, and for the treatmentof Niemann-Pick Disease type C. The invention provides specificpharmacological chaperones for β-glucocerebrosidase, which increasecytosolic trafficking, and enzymatic activity of β-glucocerebrosidase,presumably by stabilizing the enzyme.

BACKGROUND OF THE INVENTION Protein Aggregation in NeurodegenerativeDiseases

In neurons, the proteasomal and lysosomal systems work in concert tomaintain protein homeostasis by degrading damaged, misfolded or excessproteins. Many neurodegenerative diseases associated with pathologicaggregation of proteins or lipids show impaired proteasomal andlysosomal function, including Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis (ALS), Gaucherdisease, Tay Sachs, Farber, Niemann-Pick Types A, B & C, G_(M1)Gangliosidosis, G_(M2) Gangliosidosis, and MPS-I. Protein aggregation inneurons is particularly dire since neurons are unable to regeneratefollowing neurodegeneration or apoptosis that arises from neuronalstress or other causes associated with the aggregation.

Protein aggregation and lysosomes. The lysosomal system is critical inpreventing the accumulation of protein aggregates that are difficult forthe proteasomes to degrade. The importance of lysosomes in degradingprotein aggregates is supported by the numerous reports of lysosomalproteins and autophagic markers co-localizing in the same structure withprotein aggregates found in human brains (Bjørkøy et al., J Cell Biol.2005; 171(4):603-14; Tribl et al., Mol Cell Proteomics, 2005; 4(7):945-57; Wong et al., Mol Genet Metab, 2004. 82(3):192-207; Zhou, J BiolChem. 2004. 279(37): 39155-64). In particular, neutralization of theacidic compartments (lysosomes) leads to the accumulation of α-synuclein(α-syn) aggregates and exacerbates α-syn toxicity in postmitoticneuronal cells (Lee, J Neurosci, 2004; 24(8): 1888-96). Pathologic α-synaggregation is associated with Parkinson's disease and otherα-synucleinopathies. Lastly, lysosome dysfunction has been reported fortransgenic mouse models that accumulate α-syn (Meredith et al., BrainRes, 2002; 956(1):156-65; Rockenstein et al., J Neurosci Res, 2005;80(2): 247-59).

Protein aggregation and proteasomes. The proteasome is integral indegrading cytosolic proteins. Proteasomal-mediated degradation beginswith the modification of substrates by polyubiquitin chains, whichtargets proteolysis by the 26S proteasome, a multicatalytic proteasecomplex. Studies have revealed that ubiquitin is a component of many ofthe filamentous inclusion bodies characteristic of neurodegenerativediseases, suggesting activation of a common neuronal response in thistype of disease process (Lowe et al., Neuropathol Appl Neurobiol. 1990;16: 281-91). Genetic studies, including identification of mutations ingenes associated with familial Parkinson's (SNCA), and the presence ofproteinaceous cytoplasmic inclusions in spared dopaminergic nigralneurons in sporadic cases of Parkinson's, have suggested an importantrole for ubiquitin-proteasome system and aberrant protein degradation inthis disease (Betarbet et al., Exp Neural. 2005; 191 Suppl 1:S17-27).

Since it is well-established that accumulation or aggregation ofnumerous misfolded proteins and lipids in a cell, including neurons,leads to endoplasmic reticulum and cell stress, increased amounts ofpolyubiquitin, a cell “stress” protein, suggests that the proteasomalsystem is overactivated. However, an alternate theory for disruptions inneuronal homeostasis in CNS aggregation diseases is due to suppressionof the ubiquitin/proteasome pathway (Rocca et al., Molecular Biology ofthe Cell. 2001; 12: 1293-1301). Both in vivo and in vitro studies havelinked α-syn aggregation and oxidative stress, both hallmarks ofParkinson's disease, to a compromised ubiquitin-proteasome system andParkinson's disease pathogenesis. (Lev et al., Neurosci Lett. 2006;399(1-2):27-32). Specifically, exposure to reactive oxygen species (ROS)combined with proteasomal inhibition increased α-syn aggregate formationover proteasomal inhibition alone. Moreover, structural and functionaldefects in 26/20S proteasomes, with accumulation and aggregation ofpotentially cytotoxic abnormal proteins, have been identified in thesubstantia nigra pars compacta of patients with sporadic Parkinson'sdisease (McKnaught et al., Ann Neurol. 2003; 53 Suppl 3:S73-84). Inaddition, mutations in SNCA that cause the protein to misfold and resistproteasomal degradation are highly associated with familial Parkinson'sdisease. It also was shown that aggregated α-syn inhibits proteasomalfunction by interacting with S6′, a subunit of the proteasome (Snyder etal., J Mol Neurosci. 2004; 24(3):425-42). Lastly, proteasomal functionis decreased in brains of subjects with Parkinson's disease as well asin brains from individuals and animals lacking parkin, which is an E3ubiquitin ligase, and is an integral part of the ubiquitin proteasomalsystem.

Thus, a defect in protein handling by the proteasome appears to be acommon factor in sporadic and the various familial forms of Parkinson'sdisease. This same conclusion was drawn from experiments in whichcombinations of a proteasome inhibitor with agents that induce proteinmisfolding were added to a culture of dopaminergic neurons (Mytilineouet al., J Neural Transm. 2004; 111(10-11):1237-51). Preferential loss ofdopamine neurons and cell death was markedly increased when the two werecombined.

Even low levels of proteasome inhibition can lead to down regulation ofthe ubiquitin proteasome system and activation of the lysosomal systemor autophagic response (Ding et al., J Neurochem, 2003; 86(2):489-97;Iwata et al., Proc Natl Acad Sci USA, 2005; 102(37):13135-40; Butler etal., Rejuvenation Res. 2005; 8(4):227-37). It has been proposed that animbalance between endogenous ER chaperones anddamaged/denatured/misfolded proteins, leading to accumulation of thelatter, can result in senescence, inhibition of the proteasome (leadingto apoptosis), or necrosis, depending on the severity of the imbalance(Soti et al., Aging Cell. 2003; 2: 39-45). This hypothesis is referredto as the “toxic protein accumulation hypothesis.”

Lipid Defects and Neurodegenerative Diseases

It is well known that lipid accumulation is associated withneurodegeneration, as is evident from lysosomal storage disorders suchas Gaucher, Tay Sachs, Farber, Niemann-Pick Types A, B and C, G_(M1)Gangliosidosis, and MPS-I diseases. However, other lipid accumulation inneurological diseases in which there are no deficiencies in thelysosomal hydrolases also has been observed. As one example,pathological accumulations of lactosylceramide, GlcCer,G_(M2)-ganglioside, and asialo-G_(M2) are found in Niemann-Pick Type Cdisease, which is a lysosomal cholesterol storage disease that is notassociated with deficient acid sphingomyelinase due to missensemutations in the gene encoding the enzyme (Vanier et al., BrainPathology. 1998; 8: 163-74). This accumulation may be caused by othermechanisms, such as defective lipid trafficking. A healthy endosomaltrafficking system is critical to neuronal function (Buckley et al., JPhysiol, 2000; 525(Pt 1):11-9). Disruption of glycosphingolipidmetabolism, including GlcCer, impairs cellular trafficking and causescholesterol sequestration and accumulation (Pagano et al., Traffic,2000; 1(11): 807-15; Sillence et al., J Lipid Res, 2002;43(11):1837-1845; Helms et al., Traffic, 2004; 5(4):247-54). Accumulatedglycolipids form “lipid rafts” that can sequester proteins important inmaintaining normal trafficking in the endosomal system (Pagano, supra).Moreover, the defective trafficking of lipids observed in fibroblastsfrom Niemann-Pick Type C cells can be reversed by treatment with apotent inhibitor of glycosphingolipid biosynthesis (Lachmann et al.,Neurobiol Dis, 2004; 16(3):654-8), further underscoring the involvementof GlcCer and other lipids in the pathology of this disease.

Further, association with lipid rafts is required for normallocalization of α-syn to its native cellular location, the synapses(Fortin et al., J Neurosci, 2004; 24(30):6715-23). Mutations associatedwith the pathology of Parkinson's disease disrupt this association. Thuschanges in lipid raft composition that also disrupt this associationcould contribute to Parkinson's disease by impairing normal localizationand distribution of α-syn as well.

Glycosphingolipids Help to Seed Protein Aggregation

Alpha-synuclein has a high affinity for gangliosides, and wild-typeα-syn forms SDS stable complexes with gangliosides that have GlcCer attheir core (Zimran et al., N Engl J Med, 2005; 352(7):728-31). Solubleforms of both α-syn and β-amyloid protein bind strongly to G_(M1),potentially seeding aggregation (Yanagisawa et al., Neurobiol Aging,1998; 19(1 Suppl):S65-7; Yanagisawa et al., Nat Med, 1995; 1(10):1062-6;Lee et al., J Biol Chem, 2002; 277(1): 671-8; Hayashi et al., JNeurosci. 2004; May 19; 24(20):4894-902). Recently, cell basedexperiments have demonstrated mutations of the lysosomal enzymeβ-glucocerebrosidase (GCase) may increase the risk for developingParkinsons's disease (Aharon-Peretz, et al., N Engl J Med, 2004;351(19):1972-7; Goker-Alpan et al., J Med Genet, 2004; 41(12):937-40;Clark et al., Mov Disord, 2005; 20(1):100-3; Eblan et al., Mov Disord,2005; 31:31). While carriers of mutant alleles do not appear toaccumulate significant levels of glucosylceramide (histologically),subtle changes in glycosphingolipid metabolism could increase the riskfor Parkinson's disease in these individuals by, e.g., disruptingautophagic responses to normal aggregates, or inhibiting theproteasomes. In addition, patients with type 1 Gaucher disease (due toGCase deficiencies) and parkinsonism/dementia exhibited α-syn positiveinclusions in hippocampal CA2-4 neurons; one patient had brainstem-typeand cortical-type Lewy bodies, and one had marked neuronal loss ofsubstantia nigra neurons (Wong et al., Mol. Genet. Metabol. 2004; 38:192-207).

Similarly, in vitro, large unilamellar vesicles of brain lipids readilyassociated with soluble N-terminal huntingtin exon 1 fragments, thepathologic protein which accumulates in Huntington's disease, andstimulated fibrillogenesis of mutant huntingtin aggregates (Suopanki etal., J Neurochem. 2006; 96(3):870-84). Lastly, in a mouse model forSandhoff disease (G_(M2) Gangliosidosis), α-syn and β-synucleinaccumulate in neurons in addition to G_(M2) gangliosides (Suzuki et al.,Neuroreport., 2003; 14(4):551-4). Thus, manipulating gangliosidemetabolism could affect the propensity for proteins to form aggregates.

The foregoing suggests that lipid interactions in vivo could influencemisfolding of proteins and may play a significant role inneurodegenerative disease pathogenesis.

Lipid and/or Protein Accumulation and Inflammation

Inflammation has been increasingly recognized to play an important rolein the pathogenesis of Parkinson's disease. Inflammatory and immune, oreven autoimmune, stigmata, have been described in post-mortem brains ofParkinson's disease patients. Alpha-syn aggregates could activatemicroglial cells, resulting in chronic inflammation leading toneurodegeneration (Wersinger et al., Curr Med Chem. 2006;13(5):591-602).

Pharmacological Chaperones Derived from Specific Enzyme InhibitorsRescue Mutant Enzymes and Enhance Wild-Type Enzymes

It has previously been shown that the binding of small moleculeinhibitors of enzymes associated with LSDs can increase the stability ofboth mutant enzymes and the corresponding wild-type enzymes (see U.S.Pat. Nos. 6,274,597; 6,583,158; 6,589,964; 6,599,919; 6,916,829, and7,141,582 all incorporated herein by reference). In particular, it wasdiscovered that administration of small molecule derivatives of glucoseand galactose, which are specific, selective competitive inhibitors forseveral target lysosomal enzymes, effectively increased the stability ofthe enzymes in cells in vitro and, thus, increased the amount of theenzyme in the lysosome as determined by measuring enzyme activity. Thus,by increasing the amount of enzyme in the lysosome, hydrolysis of theenzyme substrates is expected to increase. The original theory behindthis strategy was as follows: since the mutant enzyme protein isunstable in the ER (Ishii et al., Biochem. Biophys. Res. Comm. 1996;220: 812-815), the enzyme protein is retarded in the normal transportpathway (ER→Golgi apparatus→endosomes→lysosome) and prematurelydegraded. Therefore, a compound which binds to and increases thestability of a mutant enzyme, may serve as a “chaperone” for the enzymeand increase the amount that can exit the ER and move to the lysosomes.In addition, because the folding and trafficking of some wild-typeproteins is incomplete, with up to 70% of some wild-type proteins beingdegraded in some instances prior to reaching their final cellularlocation, the chaperones can be used to stabilize wild-type enzymes andincrease the amount of enzyme which can exit the ER and be trafficked tothe native cellular locations.

Since some enzyme inhibitors are known to bind specifically to thecatalytic center of the enzyme (the “active site”), resulting instabilization of enzyme conformation in vitro, these inhibitors wereproposed, somewhat paradoxically, to be effective chaperones that couldhelp restore exit from the ER, trafficking to the lysosomes, andhydrolytic activity. These specific pharmacological chaperones weredesignated “active site-specific chaperones (ASSCs)” or “specificpharmacological chaperones” since they bound in the active site of theenzyme in a specific fashion and do not have a general affect on allproteins.

A method for treating Parkinson's disease in individuals havingmutations in lysosomal GCase, and hence, a reduction in GCase activity,is described in co-pending U.S. application Ser. No. 11/449,528 filed onJun. 8, 2006. Although the rescue of mutant lysosomal enzymes canreverse the pathology of certain diseases, there remains a need toreduce the pathology of diseases that do not involve particularlysosomal enzyme mutations, but for which an increase in lysosomalenzymes would be beneficial.

SUMMARY OF THE INVENTION

The present invention provides a method for the treatment or preventionof a neurological disorder in an individual, wherein the neurologicaldisorder is associated with protein and lipid aggregation within thecells of the central nervous system, by administering an effectiveamount of a specific pharmacological chaperone for GCase, but whereinthe neurological disorder is not associated with a mutant GCase.

In one embodiment, the present invention provides a method for enhancingintracellular folding and processing, and hence, activity of wild typeGCase by exposing the neurons to an effective amount of a specificpharmacological chaperone for GCase.

In on aspect of this embodiment, the neurological disorder to be treatedis an α-synucleinopathy.

In specific embodiments, the α-synucleinopathy is Parkinson's disease,Lewy Body Disease, Multiple System Atrophy, Hallervorden-Spatz disease,or Frontotemporal Dementia.

In another aspect of this embodiment, the neurological disorder to betreated is Parkinson's disease.

In another aspect of this embodiment, the neurological disorder to betreated is Niemann-Pick Type C disease (NPCD).

In one embodiment of the invention, the pharmacological chaperone is areversible inhibitor of GCase.

In a specific embodiment of this aspect of the invention, thepharmacological chaperone is an isofagomine compound, such asisofagomine or C-nonyl-isofagomine, C-benzyl isofagomine, or N-alkylderivatives of isofagomine as provided herein. In another specificembodiment, the pharmacological chaperone is a glucoimidazole compoundsuch as glucoimidazole,(5R,6R,7S,8S)-5-hydroxymethyl-2-octyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,7,8-triolor(5R,6R,7S,8S)-5-Hydroxymethyl-2-(3,3-dimethylbutyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,7,8-triol.

In a further embodiment, the increase in GCase enzymatic activity is atleast, but not limited to, 1.2,1.5, 2, 3, or 5 fold over basal levels.

In an alternative embodiment in which the neurodegenerative disorder isNiemann-Pick Type C disease, the second therapeutic agent is selectedfrom the group consisting of allopreganolone, a statin, a fenofibrate,niacin; ezetimibe, a binding resin, a specific pharmacological chaperonefor β-hexosaminidase A or acid β-galactosidase,2-N-acetylamino-isofagomine, 1,2-dideoxy-2-acetamido-nojirimycin,nagstatin, 4-epi-isofagomine, and 1-deoxygalactonojirimycin.

The present invention also provides a method of treating or preventing aneurodegenerative disorder in an individual having or at risk ofdeveloping a neurodegenerative disorder, where the individual does nothave a mutation in the gene encoding GCase, by administering to theindividual a pharmacological chaperone that binds to GCase.

The invention also provides a method for combination therapy with theGCase chaperone and other therapeutic agents for treatment of theneurological disorder. In addition, the invention provides compositionsof matter comprising an admixture of a GCase pharmacological chaperoneand another therapeutic agent. In an embodiment in which theneurological disorder is Parkinsons's Disease, parkinsonism, or LewyBody Dementia, the second therapeutic agent is selected from the groupconsisting of levodopa, an anticholinergic, a Catechol-O-methyltransferase (COMT) inhibitor, a dopamine receptor agonist, a monoamineoxidase inhibitor (MAOI), a peripheral decarboxylase inhibitor, and ananti-inflammatory.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of the patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1. FIG. 1 shows GCase enhancement in spleen, lung, brain and liverof normal C57BL6 mice treated with 200 mg/kg/day of isofagomine tartrate(IFG; AT2101) for 4 weeks.

FIG. 2. FIG. 2 shows changes in GCase activity following administrationof isofagomine tartrate to healthy volunteers.

FIG. 3A-C. FIG. 3 shows results of immunohistochemical staining forα-synuclein in the cortex of transgenic mice over-expressing α-synucleineither treated or untreated with IFG tartrate. FIG. 3A shows resultsfrom a vehicle treated control; FIGS. 3B and 3C show results from micetreated with 2 mg/kg daily for 3 months.

FIG. 4A-C. FIG. 4 shows qualitative results of immunohistochemicalstaining for α-synuclein in the hippocampus of transgenic miceover-expressing α-synuclein either treated or untreated with IFGtartrate. FIG. 4A shows results from a vehicle treated control; FIGS. 4Band 4C show results from mice treated with 2 mg/kg daily for 3 months.

FIG. 5A-D. FIG. 5 shows qualitative results of immunohistochemicalstaining for α-synuclein in samples pre-treated with Proteinase K fromthe brain of one transgenic mouse.

FIG. 6A-B. FIG. 6 shows quantitative results of immunohistochemicalstaining for α-synuclein in the cortex (A) and hippocampus (B) oftransgenic mice over-expressing α-synuclein either treated or untreatedwith IFG at the indicated concentrations for 3 months. The number ofα-synuclein positive cells per mm² was evaluated.

DETAILED DESCRIPTION

The present invention relates to the discovery that a specificpharmacological chaperone can increase the activity of wild-type GCaseto a sufficient level to inhibit, even to the point of prevention,pathology associated with the build up of aggregated proteins andsubstrate lipids. This in turn, can be used to treat neurological riskfactors, conditions, or disorders associated with the aggregation ofthose substrate proteins and lipids within the cells of the centralnervous system.

Specifically, the present invention provides a method of administeringone or more pharmacological chaperones for GCase to an individualdiagnosed, at risk, or suspected to have a neurological disorder thatcould be benefited by increased activity of GCase. Suitablepharmacological chaperones include any compound(s) which, followingadministration to an individual, will specifically bind to GCase,increase the stability and trafficking of GCase, and thereby increaseGCase activity in the lysosome, provided that the neurological diseaseor disorder is not associated with a mutation in the gene encodingGCase. Enhancing enzymatic function in the cells of the central nervoussystem, presumably as a result of a more stable intracellular form ofGCase, increases metabolism of GCase-associated peptides and GCase lipidsubstrates within the cells, which is useful in the treatment ofneurological disorders such as Parkinson's disease and Niemann-Pick TypeC disease, neither of which are associated with deficient GCaseactivity.

The invention is based, in part, on the discovery of a pharmacologicalchaperone's ability to promote significantly increased wild-type proteinactivity in humans. This phenomenon is highly specific to the proteinspecifically bound by the particular pharmacological chaperone, incontrast to methods that operate generally on expression of allproteins. The experimental results that underlie the present inventioninclude the observations that pharmacological chaperones can increaseendogenous wild-type protein activity by at least about 20-25%, in somecases by at least about 50%, and in specific embodiments by at leastabout 90%, and even 100%. This level of increase in vivo was notobserved with cells in tissue culture, and comes as a surprise, giventhe expectation that normal physiological processes would buffer theeffects of pharmacological chaperones in vivo. There was no basis toexpect that a pharmacological chaperone could increase the level ofactivity of a wild type protein in vivo in a human by at least 20-25%,and particularly, by at least about 50%. As exemplified infra,administration of isofagomine to healthy subjects resulted in adose-dependent increase in GCase activity (FIG. 2). At someconcentrations, which were readily tested using routine dose-responsetesting, the level of increase in enzyme activity increased by at least50% to up to 100%.

The present invention came about from the known link between lysosomalenzyme insufficiency and neurological disease states (e.g., Types 2 and3 Gaucher disease), and the fact that protein and/or lipid aggregationand neuronal death/degeneration is observed in many non-LSDneurodegenerative disorders, such as described earlier in the Backgroundsection. Thus, the present invention unexpectedly exploits the abilityto increase the activity of non-deficient lysosomal enzymes to increaseclearance of protein aggregates, particularly aggregates in whichproteins (or fragments thereof including monomers) are associated withlipid substrates of the lysosomal enzyme, such as, for example, SCNAwith GlcCer. Reducing the lipid substrate to which the pathologicprotein associates will likely reduce aggregation of the pathologicprotein, thereby ameliorating neurological symptoms and/or preventingneuronal death or neurodegeneration. Alternatively, the enhancement ofGCase activity may reduce the concentration of a lipid that in turnprovides a cellular environment better able to control the degradationof aggregation-prone proteins.

The invention also unexpectedly exploits the ability to increase theactivity of non-deficient lysosomal enzymes to alter the lipid profilein neurons in which there is accumulation of GlcCer, glucosylsphingosine(GlcSph; or other lipids whose levels change in response to GCaseactivity due to abnormal lipid metabolism) by a mechanism other thanreduced GCase activity, or due to abnormalities in intracellular lipidtrafficking.

The use of specific pharmacological chaperones according to the presentinvention has potential advantages over other methods of supplementingGCase, such as by administration of recombinant enzyme (ERT) or usingsubstrate reduction therapy (SRT) with, e.g., Zavesca®, since the formermust be administered directly into the brain via a catheter, and thelatter inhibits synthesis of numerous glycolipids, and not just thosethat may be beneficial to decrease for one particular neurologicaldisorder. Zavesca® has severe side effects and it use is limited topatients who cannot tolerate ERT. Therefore, these treatments are lesseffective than a treatment than can enhance hydrolase activityubiquitiously of the enzyme of choice.

Definitions Biological and Clinical

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use the invention.

The terms “neurological disorder,” or “neurodegenerative disorder” referto any central nervous system (CNS) or peripheral nervous system (PNS)disease that is associated with neuronal or glial cell defectsincluding, but not limited to, neuronal loss, neuronal degeneration,neuronal demyelination, gliosis (including macro- and micro-gliosis), orneuronal or extraneuronal accumulation of aberrant proteins or toxins(e.g., β-amyloid, or α-synuclein). The neurological disorder can bechronic or acute. Exemplary neurological disorders include but are notlimited to Gaucher disease, Parkinson's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS),Huntington's disease, Fredrich's ataxia, Mild Cognitive Impairment,Cerebral Amyloid Angiopathy, Parkinsonism Disease, Lewy Body Disease,Frontotemporal Dementia (FTD) Multiple System Atrophy (MSA), ProgressiveSupranuclear Palsy, and movement disorders (including ataxia, cerebralpalsy, choreoathetosis, dystonia, Tourette's syndrome, kernicterus) andtremor disorders, and leukodystrophies (including adrenoleukodystrophy,metachromatic leukodystrophy, Canavan disease, Alexander disease,Pelizaeus-Merzbacher disease), neuronal ceroid lipofucsinoses, ataxiatelangectasia and Rett Syndrome.

In particular, the term “α-synucleinopathy” refers to diseasesassociated with aberrant accumulation of α-syn, including but notlimited to parkinsonism, Parkinson's disease, Lewy Body Disease,Multiple System Atrophy, Hallervorden-Spatz disease, and FrontotemporalDementia.

A “neurological disorder associated with mutations in a lysosomalenzyme” refers to any neurological disorder in which mutations in one ormore lysosomal gene or genes are also present when assessed inindividuals having the neurological disorder, compared with individualsnot having the neurological disorder. In one, non-limiting example, theneurological disorder associated with Gba mutations is a lysosomalstorage disease, such as Gaucher disease, or a neurodegenerative diseaseassociated with a heterozygous Gba mutation, such as a subset ofpatients with Parkinson's disease. One aspect of the present inventionis that it relates to treatment of a neurological disorder that does notinvolve a mutation in Gba.

The term “increase in lysosomal activity” refers to increasing theamount of a lysosomal enzyme polypeptide that adopts a functionalconformation in the ER in a cell contacted with a pharmacologicalchaperone specific for a lysosomal enzyme, relative to lysosomal enzymeexpression in a cell (preferably of the same cell-type) not contactedwith the pharmacological chaperone specific for the lysosomal enzyme,therefore, increasing the rate of lipid and/or protein metabolismmediated by the lysosome in a cell, relative to the rate prior toadministration of the pharmacological chaperone(s). While an increase inactivity of a single lysosomal enzyme in the lysosome will result in anincrease in lysosomal activity, the invention advantageously providesfor increase activity of a number of lysosomal enzymes, resulting inbroad lipid and/or protein metabolism.

The aforementioned term also means increasing the efficiency oftransport of a wild-type lysosomal enzyme polypeptide from the ER to thelysosome in a cell contacted with a pharmacological chaperone specificfor the lysosomal enzyme, relative to the efficiency of transport ofendogenous wild-type lysosomal enzyme polypeptide in a cell (preferablyof the same cell-type) not contacted with the pharmacological chaperonespecific for the lysosomal enzyme.

The terms “lysosomal enzyme” or “lysosome enzyme” refer to any enzymethat functions in the lysosome. Lysosomal enzymes include, but are notlimited to α-galactosidase A; β-glucosidase; α-glucosidase;β-hexosaminidase A; β-hexosaminidase B; α-L-iduronidase;β-galactosidase; β-glucuronidase; α-glucuronidase; α-fucosidase;sulfatases; acid ceramidases; NPC1; acid sphingomyelinase; prosaposin(saposins A,B,C,D); cathepsins (A, D, H, S, Z); H(+)-ATPases; sialidase;β-galactocerebrosidase; arylsulfatase; iduronate-2-sulfatase; heparanN-sulfatase; α-N-acetylglucosaminidase; α-glucosaminideN-acetyltransferase; N-acetylglucosamine-6-sulfate sulfatase;N-acetylgalactosamine-6-sulfate sulfatase; arylsulfatase B; acidα-mannosidase; acid β-mannosidase; acid α-L-fucosidase;α-N-acetyl-neuraminidase; β-N-acetylglucosaminidase; andα-N-acetylgalactosaminidase.

The terms or “wild-type lysosomal enzyme” refer to the normal endogenouslysosomal polypeptides, and the nucleotide sequences encoding thelysosomal enzyme polypeptides, and any other nucleotide sequence thatencodes a lysosomal enzyme polypeptide (having the same functionalproperties and binding affinities as the aforementioned polypeptide),such as allelic variants in normal individuals, that have the ability toachieve a functional conformation in the ER, achieve lysosomallocalization, and exhibit wild-type activity (i.e., decrease lysosomalenzyme substrate concentrations). A wild-type lysosomal enzyme is not amutant or mutated protein or enzyme. However, the invention does notexclude the possibility that there may be more than one wild-type allelefor a lysosomal enzyme. This term thus includes polymorphisms that haveno detrimental effect on function or activity of the lysosomal enzyme.

Certain tests may evaluate attributes of a protein that may or may notcorrespond to its actual in vivo function, but nevertheless aresurrogates of protein functionality, and wild-type behavior in suchtests is an acceptable consequence of the protein rescue or enhancementtechniques of the invention. One such activity in accordance with theinvention is appropriate transport of a wild type lysosomal enzyme fromthe endoplasmic reticulum to the lysosome, its native location.

As used herein the terms “mutated protein” or “mutated enzyme” refer toproteins or enzymes translated from genes containing genetic mutationsthat result in protein sequences altered from the wild type sequencewhich have an effect on protein function or activity. In a specificembodiment, such mutations result in the inability of the protein toachieve a stable conformation under the conditions normally present inthe ER. The failure to achieve this conformation results in theseproteins being degraded, or aggregated, rather than being transportedthrough their normal pathway in the protein transport system to theirproper location within the cell. Mutations other than conformationalmutations also can result in decreased enzymatic activity or a morerapid turnover.

As used herein, the terms “pharmacological chaperone” or sometimes“specific pharmacological chaperone” (“SPC”) refer to a molecule, suchas a small molecule, protein, peptide, nucleic acid, or carbohydrate,that specifically binds to a protein and has one or more of thefollowing effects: (i) enhancing the formation of a stable molecularconformation of the protein; (ii) inducing trafficking of the proteinfrom the ER to another cellular location, preferably a native cellularlocation, i.e., preventing ER-associated degradation of the protein;(iii) preventing aggregation of misfolded proteins; and/or (iv)restoring or enhancing at least partial wild-type function and/oractivity to the protein. A compound that specifically binds to, e.g. alysosomal enzyme, means that it binds to and exerts a chaperone effecton the lysosomal enzyme and not a generic group of related or unrelatedproteins. Thus a pharmacological chaperone for a lysosomal enzyme is amolecule that binds preferentially to that lysosomal enzyme, resultingin proper folding, trafficking, non-aggregation, and activity of thatlysosomal enzyme. As used herein, this term does not refer to endogenouschaperones, such as BiP, or to non-specific agents which havedemonstrated chaperone activity against various proteins, such as DMSOor deuterated water.

In one, non-limiting embodiment, the pharmacological chaperone may be aninhibitor, or structurally similar analog thereof, of GCase. Thepharmacological chaperone may be selected from the list including, butnot limited to, isofagomine; C-nonyl-isofagomine; C-benzyl-isofagomine;N-butyl-isofagomine; N-(3-cyclohexylpropyl)-isofagomine;N-(3-phenylpropyl)-isofagomine;N-((2Z,6Z)-3,7,11-trimethyldodeca-2,6,10-trienyl)-isofagomine;N-dodecyl-isofagomine (N-dodecyl-IFG); 2-N-acetamido-isofagomine;2-hydroxy-isofagomine; 2-N-acetylamino-isofagomine; and analogs andderivatives thereof.

A “competitive inhibitor” of an enzyme can refer to a compound thatstructurally resembles the chemical structure and molecular geometry ofthe enzyme substrate to bind the enzyme in approximately the samelocation as the substrate. Thus, the inhibitor competes for the sameactive site as the substrate molecule, thus increasing the Km.Competitive inhibition is usually reversible if sufficient substratemolecules are available to displace the inhibitor, i.e., competitiveinhibitors can bind reversibly. Therefore, the amount of enzymeinhibition depends upon the inhibitor concentration, substrateconcentration, and the relative affinities of the inhibitor andsubstrate for the active site.

Non-classical competitive inhibition occurs when the inhibitor binds toa site that is remote to the active site, creating a conformationalchange in the enzyme such that the substrate can no longer bind to it.In non-classical competitive inhibition, the binding of substrate at theactive site prevents the binding of inhibitor at a separate site andvice versa. This includes allosteric inhibition.

A “non-competitive inhibitor” refers to a compound that forms strongbonds with an enzyme and may not be displaced by the addition of excesssubstrate, i.e., non-competitive inhibitors may be irreversible. Anoncompetitive inhibitor may be bonded at, near, or remote from theactive site of an enzyme or protein, and in connection with enzymes, hasno effect on the Km but decreases the Vmax.

Uncompetitive inhibition refers to a situation in which inhibitor bindsonly to the ES complex. The enzyme becomes inactive when inhibitorbinds. This differs from non-classical competitive inhibitors which canbind to the enzyme in the absence of substrate.

The term “Vmax” refers to the maximum initial velocity of anenzyme-catalyzed reaction, i.e., at saturating substrate levels.

The term “Km” is the substrate concentration required to achieve ½ Vmax.

An enzyme “enhancer” is a compound that binds to a lysosomal enzyme andincreases the enzymatic reaction rate.

The term “stabilize a lysosomal enzyme” refers to the ability of apharmacological chaperone to induce or stabilize a conformation of awild-type or functionally identical lysosomal enzyme protein. The term“functionally identical” means that while there may be minor variationsin the conformation (almost all proteins exhibit some conformationalflexibility in their physiological state), conformational flexibilitydoes not result in (1) protein aggregation, (2) elimination through theendoplasmic reticulum-associated degradation pathway, (3) impairment ofprotein function, e.g., degrading damaged, misfolded or excess proteins,and/or (4) improper transport within the cell, e.g., localization to alysosome within the cytosol, to a greater or lesser degree than that ofthe wild-type protein. Stabilization can be determined by any one of (i)increased enzyme half-life in the cell; (ii) increased levels of theenzyme in the lysosome; or (ii) increased hydrolytic activity asmeasured in cellular lysates using an artificial substrate.

As used herein, the term “efficiency of transport” refers to the abilityof a mutant protein to be transported out of the endoplasmic reticulumto its native location within the cell, cell membrane, or into theextracellular environment. The native location for a lysosomal enzyme isthe lysosome.

As used herein, the terms “patient” or “patient population” refer toindividual(s) diagnosed as having a neurological disorder or at risk ofdeveloping a neurological disorder. Diagnosing neurological disordersincludes identification of symptoms of decreased neurological function.Symptoms include, but are not limited to, tremor, trembling in hands,arms, legs, jaw, and face; rigidity, or stiffness of the limbs andtrunk; bradykinesia, or slowness of movement; postural instability, orimpaired balance and coordination; amnesia; aphasia; apraxia; agnosia;personality changes; depression; hallucinations; and delusions. Methodsof diagnosing neurological disorders are known to those skilled in theart. In one embodiment, the neurological disorder may be sporadic, withno association with a mutant genotype. In another embodiment, theneurological disorder may be due to an increased aggregation of lysosomeenzyme substrates, or other proteins or fragments thereof, within thecells of the CNS or PNS in patients who are not deficient in lysosomalhydrolases, i.e., do not have a mutation in a gene encoding a lysosomalhydrolase which results in reduced enzyme activity. Although thesepatients may have a mutation in a non-lysosomal enzyme, e.g., α-syn,which promotes aggregation. In another, non-limiting embodiment, theneurological disorder may have a genetic basis that is not addressed byany one, or combination, of chaperones used. For example, a patient mayhave a genotype consisting of a homozygous null mutation for a lysosomalenzyme in which no functional protein is produced. In this case,increasing the activity of other, non-mutated lysosomal enzymesaccording to the method of the present invention may compensate for thedeficient lysosomal enzyme.

A “responder” is an individual diagnosed with a neurological disorderassociated with a protein and lipid aggregates in cells of the centralnervous system, and treated according to the presently claimed methodwho exhibits an improvement in, amelioration, or prevention of, one ormore clinical symptoms, or improvement or reversal of one or moresurrogate clinical markers. In a specific embodiment, changes in thelevels of α-synuclein in plasma, including increases and decreasescompared to normal controls, is a surrogate marker for a positiveresponse to pharmacological chaperone therapy for GCase.

The terms “therapeutically effective dose” and “effective amount” referto the amount of the specific pharmacological chaperone that issufficient to result in a therapeutic response. A therapeutic responsemay be any response that a practitioner (e.g., a clinician) willrecognize as an effective response to the therapy, including theforegoing symptoms and surrogate clinical markers. Thus, a therapeuticresponse will generally be an amelioration of one or more symptoms of adisease or disorder, such as those described above.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce untoward reactions when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils. Water or aqueous solution saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin, 18th Edition.

The terms “about” and “approximately” generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Typical, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values. Alternatively, and particularly inbiological systems, the terms “about” and “approximately” may meanvalues that are within an order of magnitude, preferably within 5-foldand more preferably within 2-fold of a given value. Numerical quantitiesgiven herein are approximate unless stated otherwise, meaning that theterm “about” or “approximately” can be inferred when not expresslystated.

Chemical

The term “alkyl” refers to a straight or branched C₁-C₂₀ hydrocarbongroup consisting solely of carbon and hydrogen atoms, containing nounsaturation, and which is attached to the rest of the molecule by asingle bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (isopropyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl). The alkyls used hereinare preferably C₁-C₈ alkyls.

The term “alkenyl” refers to a C₂-C₂₀ aliphatic hydrocarbon groupcontaining at least one carbon-carbon double bond and which may be astraight or branched chain, e.g., ethenyl, 1-propenyl,2-propenyl(allyl), iso-propenyl, 2-methyl-1-propenyl, 1-butenyl,2-butenyl.

The term “alkynyl” refers to monovalent, unbranched or branchedhydrocarbon chain having one or more triple bonds therein. The triplebond of an alkynyl group can be unconjugated or conjugated to anotherunsaturated group. Suitable alkynyl groups include, but are not limitedto, (C₂-C₈)alkynyl groups, such as ethynyl, propynyl, butynyl, pentynyl,hexynyl, methylpropynyl, 4-methyl-1-butynyl,4-propyl-2-pentynyl-, and4-butyl-2-hexynyl. An alkynyl group can be unsubstituted or substitutedwith one or two suitable substituents.

The term “cycloalkyl” denotes an unsaturated, non-aromatic mono- ormulticyclic hydrocarbon ring system such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl. Examples of multicyclic cycloalkyl groupsinclude perhydronapththyl, adamantyl and norbornyl groups bridged cyclicgroup or sprirobicyclic groups, e.g., spiro (4,4) non-2-yl.

The term “cycloalkylalkyl” refers to a cycloalkyl as defined abovedirectly attached to an alkyl group as defined above, that results inthe creation of a stable structure such as cyclopropylmethyl,cyclobutylethyl, cyclopentylethyl.

The term “alkyl ether” refers to an alkyl group or cycloalkyl group asdefined above having at least one oxygen incorporated into the alkylchain, e.g., methyl ethyl ether, diethyl ether, tetrahydrofuran.

The term “alkyl amine” refers to an alkyl group or a cycloalkyl group asdefined above having at least one nitrogen atom, e.g., n-butyl amine andtetrahydrooxazine.

The term “aryl” refers to aromatic radicals having in the range of about6 to about 14 carbon atoms such as phenyl, naphthyl, tetrahydronapthyl,indanyl, biphenyl.

The term “arylalkyl” refers to an aryl group as defined above directlybonded to an alkyl group as defined above, e.g., —CH₂C₆H₅, and—C₂H₄C₆H₅.

The term “heterocyclic” refers to a stable 3- to 15-membered ringradical which consists of carbon atoms and from one to five heteroatomsselected from the group consisting of nitrogen, phosphorus, oxygen andsulfur. For purposes of this invention, the heterocyclic ring radicalmay be a monocyclic, bicyclic or tricyclic ring system, which mayinclude fused, bridged or Spiro ring systems, and the nitrogen,phosphorus, carbon, oxygen or sulfur atoms in the heterocyclic ringradical may be optionally oxidized to various oxidation states. Inaddition, the nitrogen atom may be optionally quaternized; and the ringradical may be partially or fully saturated (i.e., heteroaromatic orheteroaryl aromatic). Examples of such heterocyclic ring radicalsinclude, but are not limited to, azetidinyl, acridinyl, benzodioxolyl,benzodioxanyl, benzofurnyl, carbazolyl, cinnolinyl, dioxolanyl,indolizinyl, naphthyridinyl, perhydroazepinyl, phenazinyl,phenothiazinyl, phenoxazinyl, phthalazinyl, pyridyl, pteridinyl,purinyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl,tetrazoyl, imidazolyl, tetrahydroisouinolyl, piperidinyl, piperazinyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl,azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazinyl, pyrimidinyl,pyridazinyl, oxazolyl, oxazolinyl, oxasolidinyl, triazolyl, indanyl,isoxazolyl, isoxasolidinyl, morpholinyl, thiazolyl, thiazolinyl,thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl,isoindolyl, indolinyl, isoindolinyl, octahydroindolyl,octahydroisoindolyl, quinolyl, isoquinolyl, decahydroisoquinolyl,benzimidazolyl, thiadiazolyl, benzopyranyl, benzothiazolyl,benzooxazolyl, furyl, tetrahydrofurtyl, tetrahydropyranyl, thienyl,benzothienyl, thiamorpholinyl, thiamorpholinyl sulfoxide thiamorpholinylsulfone, dioxaphospholanyl, oxadiazolyl, chromanyl, isochromanyl.

The heterocyclic ring radical may be attached to the main structure atany heteroatom or carbon atom that results in the creation of a stablestructure.

The term “heteroaryl” refers to a heterocyclic ring wherein the ring isaromatic.

The term “heteroarylalkyl” refers to heteroaryl ring radical as definedabove directly bonded to alkyl group. The heteroarylalkyl radical may beattached to the main structure at any carbon atom from alkyl group thatresults in the creation of a stable structure.

The term “heterocyclyl” refers to a heterocylic ring radical as definedabove. The heterocyclyl ring radical may be attached to the mainstructure at any heteroatom or carbon atom that results in the creationof a stable structure.

The term “heterocyclylalkyl” refers to a heterocylic ring radical asdefined above directly bonded to alkyl group. The heterocyclylalkylradical may be attached to the main structure at carbon atom in thealkyl group that results in the creation of a stable structure.

The substituents in the “substituted alkyl”, “substituted alkenyl,”“substituted alkynyl,” “substituted cycloalkyl,” “substitutedcycloalkalkyl,” “substituted cyclocalkenyl,” “substituted arylalkyl,”“substituted aryl,” “substituted heterocyclic ring,” “substitutedheteroaryl ring,” “substituted heteroarylalkyl,” or substitutedheterocyclylalkyl ring,” may be the same or different with one or moreselected from the groups hydrogen, hydroxy, halogen, carboxyl, cyano,amino, nitro, oxo (═O), thio (═S), or optionally substituted groupsselected from alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl,cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocyclic ring,—COOR^(x), —C(O)R^(x), —C(S)R^(x), —C(O)NR^(x)R^(y), —C(O)ONR^(x)R^(y),—NR^(x)CONR^(y)R^(z), —N(R^(x))SOR^(y), —N(R^(x))SO₂R^(y),—(═N—N(R^(x))R^(y)), —NR^(x)C(O)OR^(y), —NR^(x)R^(y), —NR^(x)C(O)R^(y)—,—NR^(x)C(S)R^(y) —NR^(x)C(S)NR^(y)R^(z), —SONR^(x)R^(y)—,—SO₂NR^(x)R^(y)—, —OR^(x), —OR^(x)C(O)NR^(y)R^(z), —OR^(x)C(O)OR^(y)—,—OC(O)R^(x), —OC(O)NR^(x)R^(y), —R^(x)NR^(y)R^(z), —R^(x)RYR^(z),—RNR^(y)C(O)R^(z), —R^(x)OR^(y), —R^(x)C(O)OR^(y),—R^(x)C(O)NR^(y)R^(z), —R^(x)C(O)R^(x), —R^(x)OC(O)R^(y), —SR^(x),—SOR^(x), —SO₂R^(x), —ONO₂, wherein R^(x), R^(y) and R^(z) in each ofthe above groups can be hydrogen atom, substituted or unsubstitutedalkyl, haloalkyl, substituted or unsubstituted arylalkyl, substituted orunsubstituted aryl, substituted or unsubstituted cycloalkyl, substitutedor unsubstituted cycloalkalkyl substituted or unsubstituted heterocyclicring, substituted or unsubstituted heterocyclylalkyl, substituted orunsubstituted heteroaryl or substituted or unsubstitutedheteroarylalkyl.

The term “halogen” refers to radicals of fluorine, chlorine, bromine andiodine.

The term “a short flexible linker” refers to linkers with linear lengthof about 6 Å to about 12 Å, preferably about 9 Å. A short flexiblelinker comprises molecules bonded to each other, for example, but notlimited to, carbon bound to carbon and carbon bound to a heteroatom suchas nitrogen, oxygen, or sulfur, wherein the molecules bonded togethercan rotate around the axis of the bond. In a particular embodiment, theflexible linker can adopt different conformations and orientations thatcan alter the distance between molecular domains connected by the shortflexible linker.

Chaperone Therapy for Neurodegenerative Disorders

In one series of embodiments, small molecule pharmacological chaperonesincrease the stability of a non-mutant GCase in the ER, increasetrafficking to the lysosome, and increase the enzyme's half-life bystabilizing the protein in the lysosome. This strategy may result in anincrease in enzymatic activity or enzyme function in the lysosome, andhence, increased lysosomal activity. Lipid metabolism, such as GlcCermetabolism can thus be modulated by increasing GCase activity, leadingto a reduced level of GlcCer in the cell when compared to a cell notcontacted with the chaperone. As discussed above, this strategy isexpected decrease the amount of α-syn, and/or will amelioratepathological accumulation of GlcCer or any disruptive lipid imbalanceinvolving GlcCer in cells, particularly neurons.

Chaperones for GCase. There are numerous compounds that can be used,alone or in combination, as pharmacological chaperones to increaselysosomal activity by increasing the activity of GCase. As discussedabove, competitive inhibitors for GCase previously have been shown toincrease GCase activity. Accordingly, it is anticipated that these andother inhibitors of GCase may be useful for decreasing the amount ofα-syn, possibly by increasing GCase activity in the lysosome, althoughother mechanisms are possible.

Isofagomine (IFG; (3R,4R,5R)-5-(hydroxymethyl)-3,4-piperidinediol)refers to a compound having the following structure:

Isofagomine tartrate has recently been described in commonly-owned U.S.patent application Ser. No. 11/752,658, filed on May 23, 2007, and hasbeen assigned CAS number 919364-56-0. Isofagomine also may be preparedin the form of other acid addition salts made with a variety of organicand inorganic acids. Such salts include those formed with hydrogenchloride, hydrogen bromide, methanesulfonic acid, sulfuric acid, aceticacid, trifluoroacetic acid, oxalic acid, maleic acid, benzenesulfonicacid, toluenesulfonic acid and various others (e.g., nitrate, phosphate,borates, citrates, benzoates, ascorbates, salicylates and the like).Such salts can be formed as known to those skilled in the art.

N-alkyl derivatives of isofagomine are also contemplated for use in thepresent invention. Such compounds are described in U.S. Pat. No.6,046,214 to Kristiansen et al., and U.S. Pat. No. 5,844,102 to Sierkset al. In an additional embodiment, the isofagomine derivative has thefollowing Formula I:

wherein:

R is C₁₋₇haloalkyl, C₁₋₁₀alkyl, C₃₋₇alkenylalkyl, C₂₋₇alkoxyalkyl,C₁₋₇carbamoylalkyl or X—Ar¹;

X is —(CH)_(n)— or C₂-C₃ alkenylene;

n is an integer from 0-3;

Ar¹ is

wherein R₁ and R₂ are independently selected from hydrogen, halo,C₁₋₃alkyl, C₁₋₃alkoxy, amino, nitro, heteroaryl, aryl, or cyano; X and Zare independently C, N, O, or S when Y is C, N, O, or S; or X and Z areindependently C, N—R³, O or S when Y is a single bond connecting X and Zwherein R³ is C₁₋₃alkyl or hydrogen, or pharmaceutically acceptablesalts thereof.

In yet a further embodiment, the isofagomine derivative has thefollowing Formula Ia:

wherein:

R is C₁₋₁₀alkyl, C₃₋₇alkenylalkyl, C₂₋₇alkoxyalkyl, or X—Ar;

X is —(CH)_(n)— or C₂-C₃ alkenylene;

n is an integer from 0-3;

Ar is

wherein X and Z is C, N, O, or S; Y is C, N, O, S or a single bondconnecting X and Z; R₁ is a hydrogen, halo, C₁₋₃alkyl, C₁₋₃alkoxy,amino, nitro, aryl, or cyano, or pharmaceutically acceptable saltsthereof.

Specific N-alkyl derivatives include N-dodecyl isofagomine and thoseprovided in Table 1 below:

TABLE 1

Compound R 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

The compounds are named as follows:(3R,4R,5R)-5-(hydroxymethyl)-1-propylpiperidine-3,4-diol (1);(3R,4R,5R)-5-(hydroxymethyl)-1-pentylpiperidine-3,4-diol (2);(3R,4R,5R)-1-heptyl-5-(hydroxymethyl)piperidine-3,4-diol (3);(3R,4R,5R)-5-(hydroxymethyl)-1-(3-methylbut-2-enyl)piperidine-3,4-diol(4); (3R,4R,5R)-5-(hydroxymethyl)-1-(2-methoxyethyl)piperidine-3,4-diol(5); (3R,4R,5R)-1-benzyl-5-(hydroxymethyl)piperidine-3,4-diol (6);(3R,4R,5R)-5-(hydroxymethyl)-1-(2-methylbenzyl)piperidine-3,4-diol (7);(3R,4R,5R)-5-(hydroxymethyl)-1-(3-methylbenzyl)piperidine-3,4-diol (8);(3R,4R,5R)-5-(hydroxymethyl)-1-(4-methylbenzyl)piperidine-3,4-diol (9);(3R,4R,5R)-1-(4-fluorobenzyl)-5-(hydroxymethyppiperidine-3,4-diol (10);(3R,4R,5R)-5-(hydroxymethyl)-1-(4-methoxybenzyl)piperidine-3,4-diol(11); (3R,4R,5R)-1-(4-aminobenzyl)-5-(hydroxymethyl)piperidine-3,4-diol(12); (3R,4R,5R)-5-(hydroxymethyl)-1-(2-phenylethyl)piperidine-3,4-diol(13);(3R,4R,5R)-1-(biphenyl-4-ylmethyl)-5-(hydroxymethyl)piperidine-3,4-diol(14);(3R,4R,5R)-5-(hydroxymethyl)-1-((tetrahydro-2H-pyran-4-yl)methyl)piperidine-3,4-diol(15);(3R,4R,5R)-5-(hydroxymethyl)-1-(piperidin-4-ylmethyl)piperidine-3,4-diol(16);(3R,4R,5R)-1-(furan-2-ylmethyl)-5-(hydroxymethyppiperidine-3,4-diol(17);(3R,4R,5R)-5-(hydroxymethyl)-1-(thiophen-2-ylmethyl)piperidine-3,4-diol(18);(3R,4R,5R)-5-(hydroxymethyl)-1-(pyridin-4-ylmethyl)piperidine-3,4-diol(19); (3R,4R,5R)-1-cyclohexyl-5-(hydroxymethyl)piperidine-3,4-diol (20);(3R,4R,5R)-1-(cyclohexylmethyl)-5-(hydroxymethyl)piperidine-3,4-diol(21);(3R,4R,5R)-1-(2-cyclohexylethyl)-5-(hydroxymethyl)piperidine-3,4-diol(22);(3R,4R,5R)-1-(cyclopentylmethyl)-5-(hydroxymethyl)piperidine-3,4-diol(23); (3R,4R,5R)-5-(hydroxymethyl)-1-(4-methylpentyl)piperidine-3,4-diol(24); and(3R,4R,5R)-5-(hydroxymethyl)-1-(4-nitrobenzyl)piperidine-3,4-diol (25).

Additional inhibition data for these compounds towards GCase, and orsome cellular enhancement data for GCase, are provided in Table 2 below:

TABLE 2 IC₅₀ (μM) Ki (μM) EC₅₀ (μM) Compound (n = 3) (n = 3) (n = 3) 135.81 ± 6.69 14.84 ± 2.77   65.9 ± 15.6 2 979.9 ± 137  406.06 ± 56.34 266.1 ± 47.7 3 41.70 ± 3.05 17.28 ± 1.26   9.99 ± 2.56 4  4.00 ± .0351.66 ± .015  63.0 ± 14.1 5  5.99 ± 0.11 2.48 ± 0.05  60.7 ± 0.11 6200.27 ± 61.78 82.99 ± 25.6  115.3 ± 18.5 7 69.47 ± 8.18 28.79 ± 3.39 46.4 ± 8.7 8  12.7 ± 2.14 5.26 ± 0.89 11.6 ± 2.4 9 129.63 ± 14.71 53.72± 6.10  144.8 ± 2.9  10 269.80 ± 39.21 111.8 ± 16.25 70.6 ± 2.1 11 nd ndnd 12  9.36 ± 1.16 3.88 ± 0.48  7.7 ± 1.5 13  7.5 ± 0.82 3.11 ± 0.34 2.2 ± 0.1 14 nd nd nd 15 49.22 ± 5.5  20.39 ± 2.28  68.3 ± 5.7 16  1.07± 0.02 0.44 ± 0.01  6.9 ± 1.5 17 280.07 ± 62.55 116.39 ± 25.92   65.6 ±15.7 18 27.55 ± 0.49 11.42 ± 0.2  139.0 ± 27.8 19 19.35 ± 1.24 8.02 ±0.51 35.0 ± 3.6 20 nd nd nd 21 nd nd nd 22 nd nd nd 23 15.63 ± 2.2  6.48± .091 18.8 ± 2.8 24  2.13 ± 0.17 0.88 ± 0.07 30.5 ± 4.8 25 44.96 ± 3.9918.63 ± 1.65  16.4 ± 2.2 nd = not done

Methods of synthesizing isofagomine and some derivatives are well knownin the and are described in the following: Jespersen et al., Angew.Chem., Int. ed. Engl. 1994; 33: 1778-9; Dong et al., Biochem. 1996;35:2788; Lundgren et al., Diabetes. 1996; 45:S2 521; Schuster et al.,Bioorg Med Chem Lett. 1999; 9(4):615-8; Andersch et al., Chem. Eur. J2001; 7: 3744-3747; Jakobsen et al., Bioorg Med Chem. 2001; 9: 733-44;36:435; Pandy et al., Synthesis. 2001: 1263-1267; Zhou et al., Org Lett.2001;3(2):201-3; Best et al., Can. J. Chem./Rev. Can. Chim. 2002; 80(8):857-865; Huizhen et al., J. Carbohydr Chem. 2004;23: 223-238; Mehta etal., Tetrahedron Letters 2005; 41(30):5747-5751; Ouchi et al., J OrgChem. 2005;70(13):5207-14; and most recently, Meloncelli et al.,Australian Journal of Chemistry. 2006; 59(11) 827-833. Synthesis of theL stereoisomer is described in Panfil et al., J. Carbohydr Chem. 2006;25: 673-84.

Specifically, the N-alkyl isofagomine derivatives described above can bemade by routes known in the art to alkylate secondary amines, such as byeither displacement of a mesylate (from the corresponding commerciallyavailable alcohols) or by reductive amination. A brief description ofthese methods is provided below.

General Method for Alkylation

After 1 equivalent of the alcohol and 1.5 equivalent of triethylamineare dissolved in CH2Cl2, methanesulfonyl chloride (1.2 equivalent) isadded dropwise to the reaction mixture at 0° C. The reaction mixture isstirred at RT for 2 hours, and then poured into water and extracted withCH2Cl2. The solvent is removed to give the crude product which was useddirectly for next step.

3 equivalent of the crude product from the previous step and 1equivalent of 5-(hydroxymethyl)piperidine-3,4-diol and K2CO3 (5equivalents) are suspended in DMF, and the mixture is stirred and heatedat 70° C. for 3 days. The reaction mixture is filtered and the solventis removed to give a crude product. The crude product and silica gel aresuspended in MeOH.HCl solution, and stirred at RT for 30 mins. Thesolvent is removed to give a solid, which is packed to the top of silicagel column. The final compound is eluted by Ethyl Acetate/MeOH/aqammonia (9/1/0.2).

General Method for Reductive Amination

3 equivalent of aldehyde or ketone and 1 equivalent of5-(hydroxymethyl)piperidine-3,4-diol are suspended in methanol, thenNa(CN)BH3 (6 eq) is added. The reaction mixture is stirred at RT for 3days. The reaction mixture is filtered and the solvent was removed togive a crude product. The crude product and silica gel is suspended inMeOH.HCl solution, and stirred at RT for 30 mins. The solvent is removedto give a solid, which is packed to the top of silica gel column. Thefinal compound was eluted by Ethyl Acetate/MeOH/aq ammonia (9/1/0.2).

General Method for Nitro Reduction

5-(Hydroxymethyl)-1-(4-nitrobenzyl)piperidine-3,4-diol and Zinc (10 eq)are stirred in MeOH.HCl solution at RT for one hour. The solvent isremoved to give a crude product, which is purified by silica gel column.

Since these compounds specifically bind to and enhance GCase, they alsocan be used for the treatment of Gaucher disease and other neurologicaldisorders associated with mutations in GCase.

Other chaperones for GCase include glucoimidazole,polyhydroxylcycloalkylamines and derivatives, and hydroxyl piperidinederivatives, which are described in pending U.S. published applications2005/0130972 and 2005/0137223, and corresponding PCT publications WO2005/046611 and WO 2005/046612, all filed on Nov. 12, 2004 andincorporated herein by reference. Glucoimidazole and derivatives arerepresented by the following Formula II:

wherein B is selected from the group consisting of hydrogen, hydroxy,acetamino, and halogen;

R¹ and R² optionally present are short, flexible linkers linear lengthof about 6 Å to about 12 Å, preferably about 9 Å. R¹ and R² can also beindependently selected from the group consisting of C₂-C₆ substituted orunsubstituted alkyl optionally interrupted by one or more moietieschosen from the group consisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH,CONH, NHCO, NR³, O, S, S(O)_(m) and —S(O)_(m)NR³; C₂-C₆ substituted orunsubstituted alkenyl optionally interrupted by one or more moietieschosen from the group consisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH,CONH, NHCO, NR³, O, S, S(O)_(m) and —S(O)_(m)NR³; C₂-C₆ substituted orunsubstituted alkynyl optionally interrupted by one or more moietieschosen from the group consisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH,CONH, NHCO, NR³, O, S, S(O)_(m) and —S(O)_(m)NR³ , wherein m is 1 or 2,and R³ is independently selected from each occurrence from the groupsconsisting of hydrogen substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl; substituted or unsubstituted alkenyl;substituted or unsubstituted cycloalkyl, substituted or unsubstitutedcycloalkenyl; substituted or unsubstituted aryl; substituted orunsubstituted arylalkyl; substituted or unsubstituted heteroaryl;substituted or unsubstituted heterocyclic; substituted or unsubstitutedheterocyclyalkyl; substituted or unsubstituted heteroarylalkyl.

In addition, R¹-L¹ and/or R²-L² can be hydrogen.

R⁵ represents a hydrogen, hydroxy, or hydroxylmethyl;

L¹ and L² are lipophilic groups selected from the group consisting ofC₃-C₁₂ substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl substituted or unsubstituted alkynyl; substituted orunsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl;substituted or unsubstituted aryl; substituted or unsubstitutedarylalkyl; substituted or unsubstituted heteroaryl; substituted orunsubstituted heterocyclic; substituted or unsubstitutedheterocycloalkyl; substituted or unsubstituted heteroarylalkyl.

In specific embodiments, GIZ compounds include GIZ,(5R,6R,7S,8S)-5-hydroxymethyl-2-octyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,7,8-trioland (5R,6R,7S,8S)-5-Hydroxymethyl-2-(3,3-dimethylbutyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,7,8-triol.

Synthesis of the foregoing can be achieved as follows:

a.(5R,6R,7S,8S)-5-Hydroxymethyl-2-n-octyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,7,8-triol(IC₅₀ for GCase=˜0.07 nM)

A solution of(5R,6R,7S,8S)-6,7,8-Tris(benzyloxy)-5-[(benzyloxy)methyl]-2-(1-octynyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine(120 mg, 0.179 mmol) in THF/EtOH (2:1) (3 ml) is rapidly stirred withPd(OH)2/C (0.1 g) under an atmosphere of hydrogen for 14 h. Afterfiltration of the catalyst, the organic solution is concentrated on arotovap and the residue is dissolved in CH2Cl2 (10 ml). The solution iscooled in an acetone-dry ice bath and a solution of BCl3 (1.0 M) inCH2Cl2 is slowly added. The reaction mixture is warmed room temperatureand stirred for 3 hours. The reaction mixture is cooled in an ice-waterbath, water added and for 0.5 hour. Most of the solvent is removed usinga rotovap and the crude product is purified by chromatography(CHCl₃/MeOH/H₂O 64:25:4). Lyophilization from water gives the titlecompound as white foam. ¹H NMR (400 MHz, CD3OD): δ7.22 (s, 1H), 4.56 (d,1H, J=8 Hz), 4.20-4.16 (m, 1H), 3.98-3.93 (m, 2H), 3.83 (t, 1H, J=8.4Hz), 3.70 (dd, 1H, J=8.8 Hz and 10 Hz), 2.60 (t, 2H, J=7.2 Hz),1.67-1.63 (m, 2H), 1.35-1.30 (m, 10H), 0.90 (t, 3H, J=6.8 Hz). MS (ES+):313 [M+1].

b.(5R,6R,7S,8S)-6,7,8-Tris(benzyloxy)-5-[(benzyloxy)methyl]-2-(3,3-dimethylbyt-1-ynyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine(IC₅₀ for GCase=˜1.1 nM)

In a similar manner to that described in a,(5R,6R,7S,8S)-6,7,8-Tris(benzyloxy)-5-[(benzyloxy)methyl]-2-iodo-5,6,7,8-tetrahydroimidazo[1,2-a]pyridineis converted to the title compound. ¹H NMR (300 MHz, CDCl₃): δ7.41-7.14(m, 20H), 7.13 (s, 1H), 5.10 (d, 1H, J=11.4 Hz), 4.81-4.66 (m, 3H), 4.62(d, 1H, J=11.4 Hz), 4.49-4.42 (m, 3H), 4.18-4.13 (m, 1H), 4.11-4.06 (m,2H), 3.84-3.77 (m, 2H), 3.71 (dd, 1H, J=4.8 Hz and 10.5 Hz), 1.31 (s,9H). MS (ES+): 641 [M+1].

c.(5R,6R,7S,8S)-5-Hydroxymethyl-2-(3,3-dimethylbutyl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyridine-6,7,8-triol(IC₅₀ for GCase=˜0.03 nM)

In a similar manner to that described in (b),(5R,6R,7S,8S)-6,7,8-Tris(benzyloxy)-5-[(benzyloxy)methyl]-2-(3,3-dimethylbyt-1-ynyl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyridinewas converted to the title compound. ¹H NMR (400 MHz, CD₃OD): δ6.97 (s,1H), 4.41 (d, 1H, J=8 Hz), 4.09 (dd, 1H, J=2.4 Hz and 12 Hz), 3.86 (dd,1H, J=4.0 Hz and 12 Hz), 3.79-3.71 (m, 2H), 3.61 (dd, 1H, J=8.4 Hz and9.2 Hz), 2.48-2.44 (m, 2H,), 1.50-1.47 (m, 2H), 0.89 (s, 9H). MS (ES+):285 [M+1].

Polyhydroxylcycloalkylamines (PHCA) derivatives contemplated for use inthe present invention include compounds represented by the followingFormula III:

wherein B is selected from the group consisting of hydrogen, hydroxy,N-acetamino, and halogen.

R¹ is independently selected for each occurrence from the groupconsisting of hydrogen; substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,substituted or unsubstituted cycloalkyl substituted or unsubstitutedcycloalkenyl, substituted or unsubstituted aryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroaryl,substituted or unsubstituted heterocyclic, substituted or unsubstitutedheterocyclyalkyl, substituted or unsubstituted heteroarylalkyl, —C(O)R³and —S(O)_(m)R³, whereas m is 1 or 2, and R³ is independently selectedfor each occurrence from the groups consisting of hydrogen, substitutedor unsubstituted alkyl, substituted or unsubstituted alkenyl;substituted or unsubstituted alknyl; substituted or unsubstitutedcycloalkyl, substituted or unsubstituted cycloalkenyl; substituted orunsubstituted aryl; substituted or unsubstituted arylalkyl; substitutedor unsubstituted heteroaryl; substituted or unsubstituted heterocyclic;substituted or unsubstituted heterocyclyalkyl; substituted orunsubstituted heteroarylalkyl, and —C(O) attached to a C₁-C₆ substitutedor unsubstituted alkyl.

R² optionally present is a short, flexible linker linear length of about6 Å to about 12 Å, preferably, about 9 Å. R² can also be selected fromthe group consisting of C₂-C₆ substituted or unsubstituted alkyloptionally interrupted by one or more moieties chosen from the groupconsisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH, CONH, NHCO, NR³, O, S,S(O)_(m) and —S(O)_(m)NR³; C₂-C₆ substituted or unsubstituted alkenyloptionally interrupted by one or more moieties chosen from the groupconsisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH, CONH, NHCO, NR³, O, S,S(O)_(m) and —S(O)_(m)NR³; C₂-C₆ substituted or unsubstituted alkynyloptionally interrupted by one or more moieties chosen from the groupconsisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH, CONH, NHCO, NR³, O, S,S(O)_(m) and —S(O)_(m)NR³ , whereas m is 1 or 2, and R³ is independentlyselected for each occurrence from the groups consisting of hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl; substituted or unsubstituted alknyl; substituted orunsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl;substituted or unsubstituted aryl; substituted or unsubstitutedarylalkyl; substituted or unsubstituted heteroaryl; substituted orunsubstituted heterocyclic; substituted or unsubstitutedheterocyclyalkyl; substituted or unsubstituted heteroarylalkyl, and—C(O) attached to a C₁-C₆ substituted or unsubstituted alkyl; andpharmaceutically acceptable salts and prodrugs thereof.

L is a lipophilic group selected from the group consisting of C₃-C₁₂substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl; substituted orunsubstituted cycloalkyl; substituted or unsubstituted cycloalkenyl;substituted or unsubstituted aryl; substituted or unsubstitutedarylalkyl; substituted or unsubstituted heteroaryl; substituted orunsubstituted heterocyclic; substituted or unsubstitutedheterocycloalkyl; substituted or unsubstituted heteroarylalkyl.

These compounds can be made according to the methods described inpublished U.S. patent application 2005/130972.

Hydroxylpiperidine derivatives contemplated for use in the presentinvention where Gba is mutated are represented by the following FormulaIV.

wherein A represents a carbon or nitrogen;

B is a hydrogen, hydroxyl, N-acetamide or a halogen;

R¹ is a hydrogen, substituted or unsubstituted: alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkylalkyl, cycloalkenyl, aryl, arylalkyl, heteroaryl,heterocyclic, heterocyclyalkyl, or heteroarylalkyl; —C(O)R³ or—S(O)_(m)R³. Preferably, R¹ comprises H or an organic moiety having 1-12carbon atoms.

R² is an optional short, flexible linker with a linear length of fromabout 6 Å to about 12 Å. Alternatively, R² is a C₁-C₆ substituted orunsubstituted: alkyl, alkenyl, or alkynyl optionally interrupted by oneor more moieties chosen from the group consisting of NH, NHCOO, NHCONH,NHCSO, NHCSNH, CONH, NHCO, NR³, O, S, S(O)_(m) and —S(O)_(m)NR³.

R³ is of hydrogen, or a substituted or unsubstituted: alkyl, alkenyl;alknyl; cycloalkyl, cycloalkenyl; aryl; arylalkyl; heteroaryl;heterocyclic; heterocyclyalkyl; or heteroarylalkyl. Preferably, R³comprises H or an organic moiety having 1-12 carbon atoms, or morepreferably 1-6 carbon atoms.

m is 1 or 2, and

R⁵ is a hydrogen, hydroxyl, or hydroxymethyl.

L is a hydrogen, lipophilic group having 1-12 carbon atoms comprising asubstituted or unsubstituted: alkyl, alkenyl, alkynyl; cycloalkyl,cycloalkenyl; aryl; arylalkyl; heteroaryl; heterocyclic;heterocycloalkyl; or heteroarylalkyl.

In some embodiments, R²is selected from the group consisting of C₂-C₆substituted or unsubstituted alkyl optionally interrupted by one or moremoieties chosen from the group consisting of NH, NR³, and O; C₂-C₆substituted or unsubstituted alkenyl optionally interrupted by one ormore moieties chosen from the group consisting of NH, NR³ and O; C₂-C₆substituted or unsubstituted alkenyl optionally interrupted by one ormore heteroatoms chosen from the group consisting of NH, NR³ and O;C₂-C₆ substituted or unsubstituted alkenyl optionally interrupted by oneor more heteroatoms chosen from the group consisting of NH, NR³ and O.

In other embodiments, R² is chosen from the group consisting of

In other embodiments R² is not present and L is hydrogen, unsubstitutedC₁-C₁₂ alkyl, or unsubstituted C₆-C₁₂ alkyl, such as an unsubstituted C₆alkyl, unsubstituted C₇ alkyl, unsubstituted C₈ alkyl, C₉ alkyl orbenzyl.

In specific embodiments, hydroxyl piperidine compounds contemplated foruse in the invention are (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-n-hexyl-3,4-dihydroxypiperidine; (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-n-heptyl-3,4-dihydroxypiperidine; (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-n-octyl-3,4-dihydroxypiperidine; and(3R,4R,5R,6S/6R)-5-(hydroxy methyl)-6-n-nonyl-3,4-dihydroxypiperidine.

In other specific embodiments, hydroxyl piperidine compoundscontemplated for use in the present invention include but are notlimited to the following: (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-n-butyl-3,4-dihydroxypiperidine; (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-n-hexyl-3,4-dihydroxypiperidine; (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-n-heptyl-3,4-dihydroxypiperidine; (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-n-octyl-3,4-dihydroxypiperidine; (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-n-nonyl-3,4-dihydroxypiperidine; (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-benzyl-3,4-dihydroxypiperidine.

Still other chaperones for GCase are described in U.S. Pat. No.6,599,919 to Fan et al., and include calystegine A₃, calystegine A₅,calystegine B₁, calystegine B₂, calystegine B₃, calystegine B₄,calystegine C₁, N-methyl-calystegine B₂, DMDP, DAB, castanospermine,1-deoxynojirimycin, N-butyl-deoxynojirimycin, 1-deoxynojirimycinbisulfate, N-butyl-isofagomine, N-(3-cyclohexylpropyl)-isofagomine,N-(3-phenylpropyl)-isofagomine, andN-((2Z,6Z)-3,7,11-trimethyldodeca-2,6,10-trienyl)-isofagomine.

Compounds of the present invention include pharmaceutically acceptablesalts and pro-drugs of the above structures. Pharmaceutically acceptablesalts include salts derived from inorganic bases such as Li, Na, K, Ca,Mg, Fe, Cu, Zn, Mn; salts of organic bases such asN,N′-diacetylethylenediamine, glucamine, triethylamine, choline,hydroxide, dicyclohexylamine, metformin, benzylamine, trialkylamine,thiamine; chiral bases like alkylphenylamine, glycinol, phenyl glycinol,salts of natural amino acids such as glycine, alanine, valine, leucine,isoleucine, norleucine, tyrosine, cystine, cysteine, methionine,proline, hydroxy proline, histidine, omithine, lysine, arginine, serine;non-natural amino acids such as D-isomers or substituted amino acids;guanidine, substituted guanidine wherein the substituents are selectedfrom nitro, amino, alkyl, alkenyl, alkynyl, ammonium or substitutedammonium salts and aluminum salts. Salts may include acid addition saltswhere appropriate which are, sulphates, nitrates, phosphates,perchlorates, borates, hydrohalides, acetates, tartrates, maleates,citrates, succinates, palmoates, methanesulphonates, benzoates,salicylates, benzenesulfonates, ascorbates, glycerophosphates,ketoglutarates. Pharmaceutically acceptable solvates may be hydrates orcomprise other solvents of crystallization such as alcohols.

Prodrugs are compounds which are converted in vivo to active forms (see,e.g., R. B. Silverman, 1992, “The Organic Chemistry of Drug Design andDrug Action”, Academic Press, Chp. 8). Prodrugs can be used to alter thebiodistribution (e.g., to allow compounds which would not typicallyenter the reactive site of the protease) or the pharmacokinetics for aparticular compound. For example, a carboxylic acid group, can beesterified, e.g., with a methyl group or an ethyl group to yield anester. When the ester is administered to a subject, the ester iscleaved, enzymatically or non-enzymatically, reductively, oxidatively,or hydrolytically, to reveal the anionic group. An anionic group can beesterified with moieties (e.g., acyloxymethyl esters) which are cleavedto reveal an intermediate compound which subsequently decomposes toyield the active compound.

Examples of prodrugs and their uses are well known in the art (See,e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19). The prodrugs can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound with a suitable derivatizing agent. For examplehydroxy groups can be converted into esters via treatment with acarboxilic acid in the presence of a catalyst. Examples of cleavablealcohol prodrug moieties include substituted and unsubstituted, branchedor unbranched lower alkyl ester moieties, (e.g., ethyl esters), loweralkenyl esters, di-lower alkyl-amino lower-alkyl esters (e.g.,dimethylaminoethyl ester), acylamino lower alkyl esters, acyloxy loweralkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenylester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g.,with methyl, halo, or methoxy substituents) aryl and aryl-lower alkylesters, amides, lower-alkyl amides, di-lower alkyl amides, and hydroxyamides.

Formulation, Dosage, and Administration of Specific PharmacologicalChaperones

The present invention provides that the specific pharmacologicalchaperone be administered in a dosage form that permits systemicadministration, since the compounds need to cross the blood-brainbarrier to exert effects on neuronal cells. In one embodiment, thespecific pharmacological chaperone is administered as monotherapy,preferably in an oral dosage form (described further below), althoughother dosage forms are contemplated. The oral administration includesdaily administration in divided doses, or controlled-releaseformulations, or by less frequent administration of immediate- orsustained-release dosage forms. Formulations, dosage, and routes ofadministration for the specific pharmacological chaperone are detailedbelow.

Formulations

The specific pharmacological chaperone can be administered in a formsuitable for any route of administration, including e.g., orally in theform tablets or capsules or liquid, or in sterile aqueous solution forinjection. When the specific pharmacological chaperone is formulated fororal administration, the tablets or capsules can be prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets may be coated by methods well known in theart. Liquid preparations for oral administration may take the form of,for example, solutions, syrups or suspensions, or they may be presentedas a dry product for constitution with water or another suitable vehiclebefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled or sustained release of the specificpharmacological chaperone.

The pharmaceutical formulations of the specific pharmacologicalchaperone suitable for parenteral/injectable use generally includesterile aqueous solutions (where water soluble), or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be broughtabout by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, benzyl alcohol, sorbic acid, and thelike. In many cases, it will be reasonable to include isotonic agents,for example, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonosterate and gelatin.

Sterile injectable solutions are prepared by incorporating the specificpharmacological chaperone in the required amount in the appropriatesolvent with various of the other ingredients enumerated above, asrequired, followed by filter or terminal sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andthe freeze-drying technique which yield a powder of the activeingredient plus any additional desired ingredient from previouslysterile-filtered solution thereof.

The formulation can contain an excipient. Pharmaceutically acceptableexcipients which may be included in the formulation are buffers such ascitrate buffer, phosphate buffer, acetate buffer, and bicarbonatebuffer, amino acids, urea, alcohols, ascorbic acid, phospholipids;proteins, such as serum albumin, collagen, and gelatin; salts such asEDTA or EGTA, and sodium chloride; liposomes; polyvinylpyrollidone;sugars, such as dextran, mannitol, sorbitol, and glycerol; propyleneglycol and polyethylene glycol (e.g., PEG-4000, PEG-6000); glycerol;glycine or other amino acids; and lipids. Buffer systems for use withthe formulations include citrate; acetate; bicarbonate; and phosphatebuffers. Phosphate buffer is a preferred embodiment.

The formulation can also contain a non-ionic detergent. Preferrednon-ionic detergents include Polysorbate 20, Polysorbate 80, TritonX-100, Triton X-114, Nonidet P-40, Octyl α-glucoside, Octyl β-glucoside,Brij 35, Pluronic, and Tween 20.

Administration

The route of administration of the specific pharmacological chaperonemay be oral (preferably) or parenteral, including intravenous,subcutaneous, intra-arterial, intraperitoneal, ophthalmic,intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral,intradermal, intracranial, intraspinal, intraventricular, intrathecal,intracisternal, intracapsular, intrapulmonary, intranasal, transmucosal,transdermal, or via inhalation.

Administration of the above-described parenteral formulations of thespecific pharmacological chaperone may be by periodic injections of abolus of the preparation, or may be administered by intravenous orintraperitoneal administration from a reservoir which is external (e.g.,an i.v. bag) or internal (e.g., a bioerodable implant). See, e.g., U.S.Pat. Nos. 4,407,957 and 5,798,113, each incorporated herein byreference. Intrapulmonary delivery methods and apparatus are described,for example, in U.S. Pat. Nos. 5,654,007, 5,780,014, and 5,814,607, eachincorporated herein by reference. Other useful parenteral deliverysystems include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, pump delivery, encapsulated celldelivery, liposomal delivery, needle-delivered injection, needle-lessinjection, nebulizer, aerosolizer, electroporation, and transdermalpatch. Needle-less injector devices are described in U.S. Pat. Nos.5,879,327; 5,520,639; 5,846,233 and 5,704,911, the specifications ofwhich are herein incorporated by reference. Any of the formulationsdescribed above can be administered using these methods.

Subcutaneous injections have the advantages allowingself-administration, while also resulting in a prolonged plasmahalf-life as compared to intravenous administration. Furthermore, avariety of devices designed for patient convenience, such as refillableinjection pens and needle-less injection devices, may be used with theformulations of the present invention as discussed herein.

Dosage

The amount of specific pharmacological chaperone effective to rescue theendogenous mutant Lysosome enzyme can be determined on a case-by-casebasis by those skilled in the art. Pharmacokinetics and pharmacodynamicssuch as half-life (t_(1/2)), peak plasma concentration (C_(max)), timeto peak plasma concentration (t_(max)), exposure as measured by areaunder the curve (AUC), and tissue distribution for both the replacementprotein and the specific pharmacological chaperone, as well as data forspecific pharmacological chaperone/Lysosome enzyme binding (affinityconstants, association and dissociation constants, and valency), can beobtained using ordinary methods known in the art to determine compatibleamounts required to stabilize the replacement protein, withoutinhibiting its activity, and thus confer a therapeutic effect.

Data obtained from cell culture assay or animal studies may be used toformulate a therapeutic dosage range for use in humans and non-humananimals. The dosage of compounds used in therapeutic methods of thepresent invention preferably lie within a range of circulatingconcentrations that includes the ED₅₀ concentration (effective for 50%of the tested population) but with little or no toxicity. The particulardosage used in any treatment may vary within this range, depending uponfactors such as the particular dosage form employed, the route ofadministration utilized, the conditions of the individual (e.g.,patient), and so forth.

A therapeutically effective dose may be initially estimated from cellculture assays and formulated in animal models to achieve a circulatingconcentration range that will be both above the EC₅₀ observed in cellsfor a period of time, and below the observed IC₅₀ for a period of time.The EC₅₀ concentration of a compound is the concentration that achievesa half-maximal increase in enzyme activity (e.g., as determined from thecell culture assays), which the IC₅₀ is the concentration which achievehalf-maximal inhibition of enzyme activity. Appropriate dosages for usein a particular individual, for example in human patients, may then bemore accurately determined using such information as described furtherbelow.

Measures of compounds in plasma may be routinely measured in anindividual such as a patient by techniques such as high performanceliquid chromatography (HPLC) or gas chromatography.

Toxicity and therapeutic efficacy of the composition can be determinedby standard pharmaceutical procedures, for example in cell cultureassays or using experimental animals to determine the LD₅₀ and the ED₅₀.The parameters LD₅₀ and ED₅₀ are well known in the art, and refer to thedoses of a compound that is lethal to 50% of a population andtherapeutically effective in 50% of a population, respectively. The doseratio between toxic and therapeutic effects is referred to as thetherapeutic index and may be expressed as the ratio: LD₅₀/ED₅₀. Specificpharmacological chaperones that exhibit large therapeutic indices arepreferred.

Where the chaperone is an inhibitor, the optimal doses of the specificpharmacological chaperone are determined according to the amountrequired to stabilize the GCase, e.g., lysosomal enzyme, in vivo, intissue or circulation, without sustaining inhibition of the enzyme. Thiswill depend on the bioavailability of the specific pharmacologicalchaperone in tissue or in circulation, and on the pharmacokinetics andpharmacodynamics of the specific pharmacological chaperone in tissue orin circulation, for a prolonged period of time. For, the concentrationof the inhibitor can be determined by calculating the EC₅₀ and IC₅₀values of the specific chaperone for the enzyme so that the doseadministered would both (i) achieve a plasma concentration above theEC₅₀ for some time to permit maximum trafficking to the lysosome, and(ii) permit the plasma concentration to fall below the IC₅₀ for sometime once the enzyme is in the lysosome so substrate can be hydrolyzed.

This determination also will depend on pharmacokinetic factors includingthe half-life of the chaperone in blood and tissue, Tmax, and Cmax, andthe half-life of the lysosomal enzyme. In one embodiment, rationales forestimating dosing regimens for pharmacological chaperones are describedfurther in U.S. provisional patent application 60/914,288, filed on Apr.27, 2007, which is incorporated by reference herein in its entirety.This application describes “peak and trough” dosing for GCase inhibitorswhere an initial “loading dose” is given daily to maximize stabilizationof the enzyme and trafficking to the lysosome, followed by a period ofnon-daily interval dosing in which permits dissociation of the inhibitorand hydrolysis of the substrate. However, other dosing regimens also arecontemplated.

Combination Drug Therapy

The pharmacological chaperone can be used to treat patients with theneurological diseases in combination with other drugs that are also usedto treat the disorder. Conventional drug treatments for Parkinson'sdisease include but are not limited to; RNAi, and pharmacological agentssuch as levodopa, anticholinergics, COMT (Catechol-O-methyl transferase)inhibitors, dopamine receptor agonists, MAOI (monoamine oxidaseinhibitors), peripheral decarboxylase inhibitors.

The pharmacological chaperone or chaperones for GCase also can be usedto treat patients with Niemann-Pick Type C disease in combination withallopregnanolone, a low-cholesterol diet, or cholesterol-lowering agentssuch as the statins (e.g., Lipitor®); fibrates such as fenofibrate(Lipidil®); niacin; ezetimibe (Zetia®) and/or binding resins such ascholestyramine (Questran®).

In addition, the pharmacological chaperone can be used in combinationwith gene therapy. Gene therapy is contemplated both with replacementgenes such as Gba or with inhibitory RNA (siRNA) for the SNCA gene. Genetherapy is described in more detail in commonly-owned patent applicationSer. No. 10/781,356, filed on Feb. 17, 2004.

Other contemplated combination therapy includes combinations of specificpharmacological chaperones with vaccine therapy, such a vaccinecomprising α-syn and an adjuvant (Pilcher et al., Lancet Neurol. 2005;4(8):458-9), or combinations of GCase chaperones with chaperones forα-syn, such as Hsp70 or a specific pharmacologic chaperone, combinationswith anti-inflammatory agents such as ibuprofen or other NSAIDS, or withother agents that may be protective in neurodegenerative diseases suchas dextromethorphan (Li et al., FASEB J. 2005; April; 19(6):489-96),genistein (Wang et al., Neuroreport. 2005; Feb. 28; 16(3):267-70), orminoclycline (Blum et al., Neurobiol Dis. 2004; December; 17(3):359-66).

Also contemplated is combination therapy with a substrate inhibitor forGCase, such as N-butyl-deoxynojirimycin (Zavesca®).

Lastly, combinations of GCase chaperones with one or more chaperones forother lysosomal enzymes is also contemplated In one embodiment, thechaperones are administered to an individual who does not have anymutations in any of the lysosomal enzymes for which chaperones areadministered. In another embodiment, the individual has a mutation in alysosomal enzyme other than GCase and is administered a specificchaperone for that enzyme in combination with the GCase chaperone.Following is a Table which lists potential chaperones for lysosomalenzymes.

TABLE 1 LYSOSOMAL ENZYME SPECIFIC PHARMACOLOGICAL CHAPERONEα-Glucosidase 1-deoxynojirimycin (DNJ) GenBank Accession No. Y00839α-homonojirimycin castanospermine Acid β-Glucosidase(β-glucocerebrosidase) isofagomine GenBank Accession No. J03059 C-benzylisofagomine and derivatives N-alkyl(C9-12)-DNJ Glucoimidazole (andderivatives) C-alkyl-IFG (and derivatives) N-alkyl-β-valeinaminesFluphenozine calystegines A₃, B₁, B₂ and C₁ α-Galactosidase A1-deoxygalactonojirimycin (DGJ) GenBank Accession No. NM000169α-allo-homonojirimycin α-galacto-homonojirimycinβ-1-C-butyl-deoxynojirimycin calystegines A₂ and B₂ N-methylcalystegines A₂ and B₂ Acid β-Galactosidase 4-epi-isofagomine GenBankAccession No. M34423 1-deoxygalactonojirimyicn Galactocerebrosidase(Acid β-Galactosidase) 4-epi-isofagomine GenBank Accession No. D252831-deoxygalactonojirimycin Acid α-Mannosidase 1-deoxymannojirimycinGenBank Accession No. U68567 Swainsonine Mannostatin A Acidβ-Mannosidase 2-hydroxy-isofagomine GenBank Accession No. U60337 Acidα-L-fucosidase 1-deoxyfuconojirimycin GenBank Accession No. NM_000147β-homofuconojirimycin 2,5-imino-1,2,5-trideoxy-L-glucitol2,5-deoxy-2,5-imino-D-fucitol 2,5-imino-1,2,5-trideoxy-D-altritolα-N-Acetylglucosaminidase 1,2-dideoxy-2-N-acetamido-nojirimycin GenBankAccession No. U40846 α-N-Acetylgalactosaminidase1,2-dideoxy-2-N-acetamido-galactonojirimycin GenBank Accession No.M62783 β-Hexosaminidase A 2-N-acetylamino-isofagomine GenBank AccessionNo. NM_000520 1,2-dideoxy-2-acetamido-nojirimycin nagstatinβ-Hexosaminidase B 2-N-acetamido-isofagomine GenBank Accession No.NM_000521 1,2-dideoxy-2-acetamido-nojirimycin nagstatin α-L-Iduronidase1-deoxyiduronojirimycin GenBank Accession No. NM_0002032-carboxy-3,4,5-trideoxypiperidine β-Glucuronidase 6-carboxy-isofagomineGenBank Accession No. NM_000181 2-carboxy-3,4,5-trideoxypiperidineSialidase 2,6-dideoxy-2,6, imino-sialic acid GenBank Accession No.U84246 Siastatin B Iduronate sulfatase 2,5-anhydromannitol-6-sulphateGenBank Accession No. AF_011889 Acid sphingomyelinase desipramine,phosphatidylinositol-4,5-diphosphate GenBank Accession No. M59916

In one specific embodiment, Niemann-Pick Type C disease is treated witha specific pharmacological chaperone for GCase in combination with aspecific pharmacological chaperone for β-hexosaminidase A and/or aspecific pharmacological chaperone for acid β-galactosidase, since thisdisease is characterized by accumulation of G_(M2)-gangliosides andG_(M1)-gangliosides in addition to GlcCer (Vanier et al., BrainPathology. 1998; 8: 163-74).

Determining Responses to Chaperone Therapy

As indicated above, patients with neurodegenerative diseasescharacteristic neurological symptoms. For example, patients havingParkinson's disease experience tremor, rigidity, bradykinesia, andpostural imbalance. Patients having Lewy Body Dementia experience strongpsychotic symptoms (visual hallucinations) in addition to mental declinesuch as memory loss and an inability to carry out simple tasks.Observable improvements in symptoms with pharmacological chaperonetherapy, or a delay of onset of certain symptoms in patients at risk ofdeveloping a disorder, or a delay in progression of the disorder will beevidence of a favorable response to the chaperone therapy.

In addition, measurable surrogate markers also may be useful forevaluating response to chaperone therapy. For instance, someinvestigators have reported detecting higher levels of α-syn oroligomeric forms of α-syn have been detected in the plasma of patientswith Parkinson's disease (Lee et al., J Neural Transm. 2006;113(10):1435-9; El-Agnaf et al., FASEB J. 2006; 20(3):419-25), whilesome have reported decreased plasma α-syn in Parkinson's patientscompared with normal controls (Li et al., Exp Neurol. 2007;204(2):583-8).

Examples

The present invention is further described by means of the examples,presented below. The use of such examples is illustrative only and in noway limits the scope and meaning of the invention or of any exemplifiedterm. Likewise, the invention is not limited to any particular preferredembodiments described herein. Indeed, many modifications and variationsof the invention will be apparent to those skilled in the art uponreading this specification. The invention is therefore to be limitedonly by the terms of the appended claims along with the full scope ofequivalents to which the claims are entitled.

Example 1 In Vivo GCase Activity in Mice Upon Treatment with IFG

One GCase chaperone, IFG, was administered to normal mice expressingwild-type GCase and GCase activity was evaluated.

Methods

Drug administration. This Example provides information on the effects ofisofagomine, a GCase-specific chaperone on mice. IFG were administeredto the mice at 200 mg/kg/day; organs and plasma were collected 4 weeksafter initiation of the study. Ten male C57BL6 (25 g) mice per groupwere used. The drug was be given in the drinking water, therefore waterconsumption was monitored daily.

In the control group (0 mg/kg/day), the mice were dosed daily in thedrinking water (no drug) and divided into two groups. Ten animals wereeuthanized after 4 weeks of treatment, blood was collected from thedescending aorta or vena cava, and tissues were harvested and thennecropsied.

In the test group, 10 mice were dosed daily in the drinking water withan administration aim of 200 mg/kg/day.

The blood samples were drawn into lithium heparin and spun for plasma.After bleeding, the spleen, lung, brain and liver were removed andplaced into vials. The vials were put into dry ice for rapid freezing.The tissues and plasma were then analyzed for tissue levels of GCase.

Tissue preparation. Small portions of tissue were removed and added to500 μl lysis buffer (20 mM sodium citrate and 40 mM disodium hydrogenphosphate, pH 4.0, including 0.1% Triton X-100). Tissues were thenhomogenized using a microhomogenizer for a brief time, followed bycentrifugation at 10,000 rpm for 10 minutes at 4° C. Supernatants weretransferred to a new tube and used for the enzyme assay.

Tissue enzyme assay. To 2.5 μl of supernatant (in 96-well plates) wasadded 17.5 μl reaction buffer (citrate phosphate buffer, pH 4.5, noTriton X-100), and 50 μl of 4-methyl umbelliferone (4-MU)-labeledsubstrate, β-glucopyranoside, or a labeled negative controls(α-glucopyranoside or α-galacatopyranoside). Plates were incubated at37° for 1 hour, followed by the addition of 70 μl stop buffer (0.4 Mglycine-NaOH, pH 10.6). Activity of GCase was determined by measuringthe emission at 460 nm by exciting at 355 nm using a 1 second read timeper well (Victor2 multilabel counter-Wallac) Enzyme activity wasnormalized to the amount in μl of lysate added, and enzyme activity perμl of lysate was estimated. The enhancement ratio is equal to theactivity with the compound over the activity without the compound.

Results

As demonstrated in FIG. 1, GCase levels were increased following twoweeks of treatment with IFG in the liver (1), spleen (2), brain (3) andlung (4). Similar results were observed in a separate experiment where10 mice each were treated with 0, 1, 10, or 100 mg/kg/day IFG free base;wild-type GCase activity exhibited a linear dose-response increase withIFG free base.

These results support confirm that the specific pharmacologicalchaperones can increase the activity of non-mutant GCase in vivo, andparticularly in the brain.

These results support confirm that the specific pharmacologicalchaperones can increase the activity of non-mutant GCase in vivo, andparticularly in the brain.

Example 2 Administration of Multiple-Doses of IFG to Evaluate Safety,Tolerability, Pharmacokinetics and Effect on β-GlucocerebrosidaseEnzymatic Activity

It was previously shown that DGJ, a pharmacological chaperone forα-galatosidase A, another lysosomal enzyme, produced a dose-dependentincrease in α-galactosidase A activity in white blood cells of healthyvolunteers at 50 mg b.i.d. and 150 mg b.i.d.

This example describes two double-blind placebo-controlled Phase I studyof oral dosing of IFG to to evaluate the safety, tolerability,pharmacokinetics, and pharmacodynamics of IFG in healthy volunteers.

Study Design and Duration. In a first-in-human single ascending dosestudy (1a), doses of 8, 25, 75, 150 (two cohorts), and 300 mg wereadministered (6 active, 2 placebo in each cohort). In a multipleascending dose study (1b), doses of 25, 75, and 225 mg were administereddaily for seven days (6 active, 2 placebo in each cohort). In bothstudies, of the eight subjects in each group; six were randomized toreceive IFG tartrate, and two subjects received placebo. Subjects wereconfined from the evening of day-1 until 24 hrs after completion ofdosing. In the phase 1a study subjects returned at 48 hrs (PK sampling)and 7 days (safety follow-up) following dosing. In the phase 1b studysubjects returned at 48h (PD sampling), 7 days (PD sampling and safetyfollow-up), and 14 days (PD sampling) following the last dose

Study Population. Subjects were healthy male and female volunteersbetween 19 and 55 years of age (inclusive) consisting of members of thecommunity at large.

Safety and Tolerability Assessments. Safety was determined by evaluatingvital signs, laboratory parameters (serum chemistry, hematology, andurinalysis), physical examination and by recording adverse events duringthe Treatment Period.

Pharmacokinetic Sampling. Blood samples (10 mL each) were collected inblood collection tubes containing EDTA before dosing was determined atregular intervals for the phas 1a during 48 hr following dosing. In thephase 1b study a full IFG tartrate pharmacokinetic profile wasdetermined for 24 hr following the first dose, Cmin values were obtainedpre-dose on day 6 and 7, and another full profile was determined for 24hr following the final, day 7, dose.

GCase Enzymatic Activity SamplingGCase activity was determined pre-doseon days 1, 3, 5, 7, 9, 14, and 21. Blood samples were cooled in an icebath and centrifuged under refrigeration as soon as possible. Plasmasamples were divided into two aliquots and stored at 20±10° C. pendingassay. At the end of the study, all samples were transferred to MDSPharma Services Analytical Laboratories (Lincoln) for analysis. Thecomplete urine output was collected from each subject for analysis ofIFG to determine renal clearance for the first 12 hours afteradiministration of IFG tartrate on days 1 and 7.

Statistical Analysis. Safety data including laboratory evaluations,physical exams, adverse events, ECG monitoring and vital signsassessments were summarized by treatment group and point of time ofcollection. Descriptive statistics (arithmetic mean, standard deviation,median, minimum and maximum) were calculated for quantitative safetydata as well as for the difference to baseline. Frequency counts werecompiled for classification of qualitative safety data.

Results

Pharmacokinetics. In both studies, isofagomine tartrate was generallywell tolerated at all doses and treatment-emergent adverse events inboth studies were mostly mild. No serious adverse events occurred.

Isofagomine tartrate showed good systemic exposure via the oral route.In the single-dose study, plasma AUC and Cmax values were linearlycorrelated with administered dose. Mean plasma levels peaked at 3.4 hr.(SEM: 0.6 hr.) and the plasma elimination half-life was 14 hr. (SEM: 2hr.). In the multiple-dose study, after 7 days of oral administration,the pharmacokinetic behavior was found to be linear with dose, with nounexpected accumulation of isofagomine tartrate. Tmax and half-lifevalues were similar to those observed in the single-dose study.

After repeated doses of 25 to 225 mg of IFG were administered to healthymale and female subjects, the mean half-lives ranged from 5.14 to 19.9hours. Minimal accumulation of 1FG was observed after repeated doses,based on AUC and Cmax comparisons on Day 1 and Day 7. There were noobservable sex differences in any pharmacokinetic parameters evaluated.

The adverse events most frequently reported by healthy adult subjectswho received IFG included contact dermatitis, headache, nausea,increased serum bilirubin, dizziness, scab, ocular hyperaemia, andpuncture site pain. No serious adverse events occurred and no subjectdiscontinued treatment due to an adverse event.

GCase Activity. In all subjects receiving IFG, there was a markedincrease in GCase levels during the treatment period, followed by adecrease upon removal of the drug and a return to near baseline levelsby day 21, two weeks after the last dose of IFG. The increases in enzymelevels were dose-related, reaching approximately 3.5-fold above baseline(FIG. 2). These results indicate that orally administered IFG has thepotential to increase the levels of its intended target, GCase, in vivoin humans.

Example 3 Evaluation of IFG in Transgenic Mice Overexpressingα-Synuclein

This example describes results from administration of IFG to transgenicmice over-expressing α-synuclein. Neuronal expression of wild-type humanα-synuclein resulted in progresssive accumulation of α-synuclein, andubiquinated immunoreactive inclusions, including nuclear andcytoplasmic, in neurons in the neocortex, CA3 hippocampus, andsubstantia nigra by age two months (Masliah et al., Science. 2000; 287:1269). Based on the ability of IFG to increase the activity of wild-typeGCse in mice and humans, it was anticipated that increasing GCase inthese mice might compensate for the over-expressed α-synuclein andreduce or eliminate inclusions.

Methods

Mice. 48 transgenic animals (male and female) were allocated to 6 groups(n=8) at an age of 5 to 6 weeks concerning baseline and 3 to 4 weeks,respectively concerning all other treatment groups at treatment start.One group of transgenic animals served as baseline group (5 weeks old)and was sacrificed untreated on day 0. One group of transgenic animalswas treated with vehicle.

Treatment. Mice (ages 3-4 weeks) were treated once daily (orally) ateither 2 mg/kg; 20 mg/kg; or 200 mg/kg of IFG tartrate for 3 months. Onegroup also was treated once every other day at 20 mg/kg.

Tissue preparation. Animals of the baseline group were sacrificed at anage of 5 weeks. All other animals were sacrificed at the end of the 3months treatment period and blood for preparation of serum andmacrophages as well as lung, brain and CSF were extracted. Therefore,all mice were sedated by standard inhalation anesthesia (Isofluran,Baxter). Cerebrospinal fluid was obtained by blunt dissection andexposure of the foramen magnum. Upon exposure, a Pasteur pipette wasinserted to the approximate depth of 0.3-1 mm into the foramen magnum.CSF was collected by suctioning and capillary action until flow fullyceases. CSF was immediately frozen and kept at −80° C.

After CSF sampling, each mouse was placed in dorsal recumbence, thethorax was opened and a 26-gauge needle attached to a 1 cc syringe wasinserted into the right cardiac ventricular chamber. Light suction wasapplied to the needle. Blood was separated into 2 parts. One part wascollected in 3.8% sodium citrate to obtain plasma and macrophages, onepart to obtain serum. Mice were transcardially perfused withphysiological (0.9%) saline and lung tissue as well as brain was rapidlyremoved. The lungs were rinsed in cold PBS (outside only) to removeblood and then they were quick frozen.

The brains were removed and hemisected. The right hemispheres of allmice were immersion fixed in freshly produced 4% paraformaldehyde/PBS(pH 7.4) for one hour at room temperature. Thereafter brains weretransferred to a 15% sucrose PBS solution for 24 hours to ensurecryoprotection. On the next day brains were frozen in liquid isopentaneand stored at −80° C. until used for histological investigations. Todetermine the effects of the IFG-tartrate treatment from 8 animals pergroup 10 μm-thick cryosections were cut for determination ofalpha-synuclein pathology. All organs and tissues mentioned were sampledand from specific brain regions i.e. hippocampus, midbrain, frontalcortex and striatum of one hemibrain were extracted and frozen. Theother brain half was processed for histological evaluation.

Staining. For evaluation of the number of α-synuclein positive cells andLewy body like inclusions an immunohistochemical staining was carriedout using the monoclonal human alpha-synuclein specific antibody(Alexis®; Cat #804-258-L001), dilution 1:5, detected with secondary Cy2antibody (Jackson Immunoresearch®). The number of alpha-synucleinpositive cells in five different layers, one slice per layer, wascounted for evaluation in the whole hippocampus and cortex separately.

Briefly, samples were washed for 10 min with PBS at room temperature,followed by fixing for 30 min at room temperature with 4%paraformaldehyde, followed by washing 2 times for 5 min each with PBS atroom temperature. For Proteinase K-treated sections, sections wereincubated in PBS containing 10 μg/ml proteinase-K for 10 minutes at roomtemperature. Next, samples were incubated for 15 min at room temperaturewith blocking solution to block endogenous peroxidase, followed by twomore 5 min washes with PBS. Samples were then incubated for 60 min atroom temperature with a blocking reagent to block non-specific binding,followed by two more 5 min washes with PBS. Samples were then incubatedwith M.O.M. diluent for 5 min at room temperature, followed byincubation with the primary antibody (dilution 1:5 in M.O.M. diluent)for 60 min with at room temperature.

After an hour, samples were washed 2 times for 5 min with PBS at roomtemperature, and incubated for another 60 min at room temperature withblocking reagent. Following two washes, samples were incubated with withsecondary Ab Cy 2-Goat Anti-Rat (Dilution 1:200 in M.O.M.) for 60minutes at room temperature, in the absence of light. Samples were thenwashed for 5 min with PBS, for 5 min with sterile, molecular-biologygrade water, and covered with polyvinyl alcohol.

Determination of Brain α-Synuclein Pathology. For evaluation of theα-synuclein immunoreactivity, specialized image analysis software (ImagePro Plus, version 4.5.1.29) was used. Each fluorescence image wasrecorded using the same exposure time set to 400 ms. Up to 100 singleimages with 100-fold magnification each were assembled to one image(real size about 3×1 m), assuring a high pixel resolution for the IRcount. All assembled images were contrasted manually and for detectionin all measurements, the same intensity based threshold was used. A sizerestriction to a minimal size of 30 μm² was set to be sure to count asingle cell in the very acuity layer and only once among the adjacentlayers. During the macro based rating procedure the outlines of theobject counts were saved.

In a further step, all measured objects were extracted from the original(contrast free) image using the saved outlines and assembled accordingto object size in a sorted object image. The sorted object images werere-evaluated using a roundness restriction (lower limit: 1; upper limit:1.5) to part α-synuclein positive cells from bias objects. Theseautomatic object counts were visually controlled and the count manuallycorrected by adding all explicit cells not being round enough or notseparable from background in the spread sheet, leading to the ultimatecell count.

The following parameters were evaluated and calculated:

-   -   measurement area of the cortex and the hippocampus in each        slice; and    -   number of IR positive cells per measurement area of the specific        brain regions hippocampus and cortex

Results

Qualitative. Differences between treated and untreated mice werequalitatively visible in different degrees from strongly decreasedbackground and decreased α-synuclein positive cell numbers in some ofthe IFG-tartrate (2 and 20 mg/kg b.w. daily) treated animals, tobackground reduction without decreased immunoreactive cell numbers, ascompared to the pathology of control animals (FIGS. 3A-C, cortex; FIGS.4A-C, hippocampus). The intensity of background in human α-synucleinoverexpressing mice derives from intracellular α-synuclein in neuriticand dendritic processes as well as synapses, therefore a reduction ofbackground is equatable to a reduction of protein expression in theseneuronal structures.

This is the first demonstration that this mouse model developsaggregated α-synuclein, as evidenced by staining of sections from amouse treated with a vehicle control (FIG. 5A). Pre-treatment ofsections with Proteinase-K prior to staining for α-synuclein alsoreveals the presence of aggregated α-synuclein by digestion of monomericα-synuclein (FIG. 5C). Pre-treatment of the mouse with IFG appears toreduce the α-synuclein aggregation. The image represents analysis ofonly a single animal to date that responded to 20mg/kg IFG-tartrate inthe absence or presence of pre-treatment with Proteinase K (FIGS. 5B andD, respectively). Since α-synuclein aggregates are resistant toProteinase K digestion, the pre-treatment reveals that aggregates ofα-synuclein accumulate in this animal model over the time frameinvestigated, and that treatment with IFG-tartrate in the 3 months priorto sacrifice prevents the accumulation of these α-synuclein aggregates.

Quantitative. The treatment with IFG-tartrate led to decreasedα-synuclein pathology in both measured brain regions, however the effectwas more pronounced in the hippocampus (FIG. 5B) and did not reachsignificance level in the cortex (FIG. 6A). The dose response wasclearly visible in the hippocampus and potentially in the cortex. Thelowest dose, 2 mg IFG-tartrate/kg, was the most effective and reducedthe number of immunoreactive cells in the hippocampus significantlyversus vehicle-treated controls (p<0.05) (FIG. 6B). Higher dosage didnot lead to statistical significant reduction of α-synucleinpathology.There was no difference between a daily or every other day treatmentwith 20 mg IFG-tartrate, and the mean was on the same level. However, itmust be stated that the 20 mg dose was not effective versus controls andtherefore the result that each or every other day treatment has the sameeffectiveness may not be accurate.

The evaluations in the baseline group led to following results: First,at the age of five weeks there was variation in individual miceregarding appearance of α-synuclein filled cells in the hippocampus,leading to variation of hippocampal pathology and high statisticalstandard deviation. In addition, the number of immunoreactive cells inthe cortex was consistently low at this age. Therefore, the increase ofpathology during ageing was significant versus all investigated groupsin the cortex, but did not reach significance level in the hippocampusdue to the individual differences in this region in the baseline group.

After averaging both regions to an individual mean, equal to increasingthe total investigated volume per animal, all groups had significantlyhigher α-synuclein positive cell load versus baseline (p<0.01) exceptmice treated with most effective dose, 2 mg IFG-tartrate (p>0.05). Thisallows the conclusion that the dosage of 2 mg IFG-tartrate/kg preventedmice from the age-accompanied increase of pathology.

These data support that increasing GCase may decrease α-synuclein levelsand prevent aggregation of α-synuclein monomers into oligomericaggregates. Further supporting for this finding comes from recentresults in which elevated levels of plasma α-syn (as detected by ELISA)were observed in patients having Gaucher disease (i.e., having decreasedGCase activity) compared to normal controls (p=0.027). Plasma fromfifty-three males and females with Gaucher disease (including one malewith Type 3) were evaluated.

Example 4 Accumulation of α-Synuclein and the Development of aParkinsonism Behavioral Phenotype in the Brains of Mice with ReducedGCase Activity

This example describes results from immunohistochemical analyses ofalpha-synuclein in the brains of transgenic mutant knock-in mice withreduced GCase activity and V394L homozygous knock-in mice treated withconduritol-β-epoxide (CBE) an irreversible inhibitor of GCase.Transgenic mouse models which have reduced GCase activities andaccumulate glucosylceramide (Sun et al 2005, Journal of lipid research)in the brain, also accumulated ubiquitinated and aggregated α-synulcein.A transgenic mouse model for Gaucher disease has been developed which donot accumulate glucosylceramide or α-synuclein in the brain, but whichcould be induced to accumulate glucosylceramide and α-synuclein bytreatment with conduritol-J3-epoxide (CBE), an irreversible inhibitor ofGCase.

Methods

Mice. Mice homozygous for the point mutations D409H or V394L andexpressing a low level of saposin C in a prosaposin knock-outbackground, were analyzed for accumulation of α-synuclein in the brain.These mice have been reported to accumulate glucosylceramide in thebrain. Mice homozygous for the point mutations V394L only, were analyzedfor α-synuclein accumulation with and without CBE treatment. These micedo not accumulate glucosylceramide in the brain unless treated with CBE.Mice were administered CBE daily by interperitonally injection for 30consequitive days (500 μM calculated from the total weight of the mouse)and brains were analyzed by immunohistochemistry for accumulation ofα-synuclein.

Tissue preparation and staining. Brain tissue was frozen and fixed forimmunohistochemistry with 4% paraformaldehyde and serial sections wereco-labelled with anti-human α-synuclein (ab1903; Abcam, Cambridge,Mass.) and rabbit anti-ubiquitin (ab7780; Abcam, Cambridge, Mass.).

Results

Serieal sections from the brains of mice having a combined Gba mutation(D409H or V394L) with reduced expression of the GCase-activating proteinprosaposin C were stained for α-synuclein and ubiquitin. In 10-wk oldmice, a significant number of α-synuclein aggregates were observed inhippocampus, basal ganglia (caudate, putamen, substantia nigra,subthalamic nucleus), brain stem, and some cortical and cerebellarregions. Ubiquitinated aggregates were also found in these regions andsome co-localized with α-synuclein. However, α-synuclein aggregates werenot observed in V394L mice (with normal prosaposin). Treatment of theV394L mice with CBE for 30 days, however, resulted in accumulation ofα-synuclein in the brain. Combined, these results suggest that reducingGCase activity and increasing glucosylceramide levels in the brain maylead to an increase in α-synuclein accumulation.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

1-34. (canceled)
 35. A method for treating in an individual having or atrisk of developing an alpha-synucleinopathy, wherein the individual doesnot have a mutation in the gene encoding beta-glucocerebrosidase,comprising administering to the individual (i) a pharmacologicalchaperone that binds to beta-glucocerebrosidase in an amount effectiveto treat the alpha-synucleinopathy and (ii) a second therapeutic agent.36. The method of claim 35, wherein the second therapeutic agent is asubstrate inhibitor for GCase.
 37. The method of claim 36, wherein thesubstrate inhibitor is N-butyl-deoxynojirimycin.
 38. The method of claim35, wherein the second therapeutic agent is an anti-inflammatory agent.39. The method of claim 38, wherein the anti-inflammatory agent isibuprofen or other NSAID.
 40. The method of claim 35, wherein the secondtherapeutic agent is dextromethorphan, genistein, or minocycline. 41.The method of claim 35, wherein the second therapeutic agent is selectedfrom the group consisting of RNAi, levodopa, an anticholinergic, aCatechol-O-methyl transferase inhibitor, a dopamine receptor agonist, amonoamine oxidase inhibitor, and a peripheral decarboxylase inhibitor.42. The method of claim 35, wherein the second therapeutic agentcomprises inhibitory RNA (siRNA) for the SNCA gene.
 43. The method ofclaim 35, wherein the second therapeutic agent comprises gene therapyfor the GBA gene.
 44. The method of claim 35, wherein the secondtherapeutic agent is a pharmacological chaperone for another lysosomalenzyme.
 45. The method of claim 35, wherein the second therapeutic agentis a vaccine comprising alpha-syn and an adjuvant.
 46. The method ofclaim 35, wherein the second therapeutic agent is a chaperone foralpha-syn.
 47. The method of claim 46, wherein the chaperone foralpha-syn is Hsp70.
 48. The method of claim 35, wherein thepharmacological chaperone is a competitive inhibitor ofβ-glucocerebrosidase.
 49. The method of claim 35, wherein thepharmacological chaperone is an isofagomine compound.
 50. The method ofclaim 35, wherein the pharmacological chaperone is isofagomine tartrate.51. The method of claim 35, wherein the α-synucleinopathy is selectedfrom the group consisting of Parkinson's disease, Lewy Body Disease,Multiple System Atrophy, Hallervorden-Spatz disease, and FrontotemporalDementia.
 52. The method of claim 35, wherein the α-synucleinopathy isParkinson's disease.
 53. The method of claim 35, wherein the effectiveamount of the pharmacological chaperone increases the activity ofβ-glucocerebrosidase.
 54. The method of claim 35, wherein the effectiveamount of the pharmacological chaperone modulates levels of α-synucleinin plasma of the individual.